Vaccine for immunocompromised hosts

ABSTRACT

The invention provides peptides derived from a ubiquitous protein, and nucleic acids encoding such peptides. The invention extends to various uses of these peptides and nucleic acids, for example, as antigens for use in vaccines per se and in the generation of antibodies for use in therapeutic drugs for the prevention, amelioration or treatment of infections caused by sepsis-inducing bacteria. The invention particularly benefits immunocompromised hosts such as neonates, babies, children, women of fertile age, pregnant women, foetuses, the elderly and diabetics.

FIELD OF THE INVENTION

The present invention relates to diseases, disorders and conditionscaused by sepsis-inducing bacteria and particularly, although notexclusively, to the treatment and prevention of sepsis andsepsis-related pathologies. The invention extends to novel peptides andtheir encoding nucleic acids, and to the use of these peptides to createvaccines for the prevention of infection by sepsis-inducing bacteria byimmunisation or by passive antibody transfer. This inventionparticularly benefits immunocompromised hosts such as neonates, babies,children, women of fertile age, pregnant women, foetuses, the elderlyand diabetics.

BACKGROUND OF THE INVENTION

Sepsis is a major cause of neonatal morbidity and mortality. Accordingto the World Health Organization (WHO), approximately one million deathsper year are caused by neonatal sepsis [1-3]. In addition, 30-50% of thesurviving neonates suffer from long term sequelae such as cognitiveimpairments, seizures or deafness [4].

Neonatal infections can occur before birth (in utero), during labour orafter birth. In utero infections are caused by the ascending ofcommensal bacteria from the mother's genital tract into the amnioticfluid [1]. Infections that occur during labour are caused by theaspiration of microbes colonising the mucosa from the mother's genitaltract. In both causes, up to 87% of infections are caused by Group BStreptococcus (GBS; also known as Streptococcus agalactiae (S.agalactiae)), Escherichia coli (E. coli) and Klebsiella spp. [5-7].Although vertical transmission of bacteria may also be the cause ofinfections occurring after birth, most of these infections are caused byStaphylococcus spp., Streptococcus pneumoniae (S. pneumoniae) orPseudomonas spp. [2,3,7-12].

In utero infections are also an important cause of preterm births. Infact, 50-80% of preterm births at <32 weeks of gestation are caused byascending bacterial infections [9,13-17].

The current treatment available for neonatal sepsis is based only onantibiotic administration. However, whilst there have been dramaticdeclines in GBS infections since the implementation of intrapartumantibiotic prophylaxis, the increase of host resistance to the usedantibiotics, as well as its questionable use in pregnant humans,highlights the need for an alternative prophylactic strategy.Immunotherapies adopted for neonatal sepsis were, however, so far,completely delusive.

The uniqueness of the neonate's immune system is based on differentcircumstances. Importantly, birth represents a dramatic passage from thealmost sterile environment offered by the mother's womb into a “hostile”antigen- and pathogen-rich outside world, to which the baby's immunesystem needs to learn to be tolerant. In that sense, the first months ofa baby's life are characterised by an active immune-tolerant status inorder to control excessive responses to new antigens, which, in turn,may increase risk of infection. On the other hand, due to the limitedexposure to antigens in utero and the well-described defects in neonataladaptive immunity, newborns must rely on their innate immune systems forprotection to infections. In fact, neutropenia (a granulocyte disordercharacterised by an abnormally low number of neutrophils), is stronglyassociated with severe sepsis [13,18-23].

Although neutropenia is usually explained by the immaturity of theneonate immune system, the inventors have previously described thatneonatal susceptibility to GBS infections is related to neonatalpredisposition to produce high amounts of interleukin-10 (IL-10), animmunosuppressive molecule, rapidly after bacterial challenge [24].Their results demonstrated that this early immunosuppression caused byIL-10 production, and not the immaturity of the neonatal immune system,was the main reason for the neutropenia observed in GBS infections [24].Moreover, they have identified extracellular glyceraldehyde-3-phosphatedehydrogenase (GAPDH) as the bacterial factor responsible for this earlyIL-10 production [24,25]. They have used recombinant GAPDH (rGAPDH) in amaternal vaccine and shown it to be highly effective in protecting theprogeny against lethal GBS infections [24]. They have shown that thisprotection can also be obtained either by antibody neutralisation of GBSGAPDH or by blocking IL-10 binding to its receptor [24].

Neisseria meningitidis (N. meningitidis), often referred to asmeningococcus, is a major cause of life-threatening sepsis, meningitisand other forms of meningococcal disease in babies and infants, but itis rarely found during the neonatal period. It is also the main cause ofbacterial meningitis in children and young adults. Serotype distributionvaries markedly around the world. In the US, for example, serogroup B isthe predominant cause of disease and mortality, followed by serogroup C;serogroup A, however, has been the most prevalent in Africa and Asia.The multiple subtypes have hindered development of a universal vaccinefor meningococcal disease; a small number of vaccines are, however,available against individual, or in one case, two, serogroups.

Along with neonates, babies, infants and children, immunocompromisedadults such as the elderly are also very susceptible to bacterialinfections and sepsis [26]. Pneumonia, bacteremia and sepsis are veryfrequent in the elderly and constitute an important cause of mortalityand morbidity. These infections are generally mixed, frequently due toanaerobes, S. pneumoniae, Staphylococcus aureus (S. aureus) andHaemophilus influenzae, although gram-negative intestinal bacteria(Klebsiella pneumoniae (K. pneumoniae) and other Enterobacteriaceae),Pseudomonas aeroginosa (P. aeruginosa) (in bedridden patients) and GBScan also be a cause [26].

Thus, the mortality associated with severe sepsis and septic shockobserved in intensive care units is around 30% [27]. Importantly, theincidence of immunocompromised patients has increased steadily for thelast 20 years [28] and immunodeficiency is a prognostic factor that ismore and more often identified as associated with the increasedmortality attributed to severe sepsis and septic shock [29].

Also, diabetic patients have increased susceptibility to invasiveinfections caused by Staphylococcus spp. and GBS [30,31]. In East Asia,diabetes is a well-known risk factor for liver abscess caused by K.pneumoniae [32].

The data found in the literature therefore indicate that there is asmall number of microbial pathogens that are consistently associatedwith sepsis across these patient groups.

SUMMARY OF THE INVENTION

Having focused their efforts on GAPDH, the inventors have nowdemonstrated that this enzyme is an extracellular virulence factor ofall relevant sepsis-inducing bacteria. The inventors believe that theyare the first ever to identify a novel array of GAPDH-derived peptidesthat are capable of eliciting antibodies that are specific forbacterial, rather than human, GAPDH. As described and exemplified fullyherein, these novel peptides are extremely useful for the generation ofvaccines for preventing infectious diseases caused by sepsis-inducingbacteria, particularly in immunocompromised hosts such as neonates,babies, children, women of fertile age, pregnant women, foetuses, theelderly and diabetics. In addition, the elicited antibodies can be usedas therapeutic agents for treating existing infections, particularly inthese patient populations.

Hence, according to a first aspect of the invention, there is providedan isolated peptide that has at least 90% amino acid sequence identitywith a peptide found within GAPDH of one or more sepsis-inducingbacteria, and has less than 10% amino acid sequence identity with apeptide found within human GAPDH, or a functional fragment or functionalvariant thereof.

The inventors were aware from their previous work that susceptibility tosepsis caused by GBS is strongly associated with the host's tendency toproduce high levels of IL-10 upon contact with bacterial GAPDH [24].However, there was no reason for the inventors to consider or suspectthe same to be true for other sepsis-inducing bacteria. Indeed, while itwas known that other sepsis-inducing bacteria possess GAPDH (becausethis enzyme is ubiquitous), it was not known or expected that GAPDH fromthe other sepsis-inducing bacteria causes IL-10 to be produced by hostcells. Thus, the discovery by the inventors that GAPDH is anextracellular virulence factor of all relevant sepsis-inducing bacteriawas completely surprising.

Furthermore, there is currently no vaccine that efficiently protectsneonates and foetuses against infections caused by each of the genera ofsepsis-inducing bacteria individually. In addition, the preventive ortherapeutic strategies used to combat sepsis in immunocompromised adultssuch as the elderly and diabetics are far from effective. As explainedherein, antibiotics only resolve part of the problem, since in the casesof late-onset sepsis they are often administered too late, and areineffective in preventing sepsis-associated morbidity. Moreover,antibiotics are not able to prevent in utero infections. The peptides,fragments and variants of the first aspect thus have significant utilityin creating a variety of useful and much-needed vaccines, particularlyfor these patient populations.

A vaccine is the most cost-effective treatment for infectious diseases,even more when the same vaccine could prevent infections caused bydifferent human pathogens in different patient groups. The presentinvention relates to the prevention, treatment and amelioration ofinfectious diseases caused by sepsis-inducing bacteria, particularlyGBS, E. coli, Staphylococcus spp., S. pneumoniae, K. pneumoniae, N.meningitidis and/or Pseudomonas spp.

DETAILED DESCRIPTION

The invention described herein is based upon the inventors' surprisingdiscovery that the susceptibility to bacterial sepsis is stronglyassociated with the host's tendency to produce high levels of IL-10 uponcontact with bacterial GAPDH, for all sepsis-inducing bacteria.

Susceptibility to sepsis is very frequent in different risk groups.Nevertheless, the microbial pathogens that are associated with sepsisare highly conserved in the different groups of susceptible hosts. Themost relevant bacteria associated with sepsis in humans are GBS, E.coli, Staphylococcus spp., S. pneumoniae, K. pneumoniae, Pseudomonasspp. and N. meningitidis, and the inventors have surprisingly found thatall of these bacteria secrete GAPDH.

As such, the sepsis-inducing bacteria may preferably be selected from agroup of bacteria consisting of GBS, E. coli, Staphylococcus spp., S.pneumoniae, K. pneumoniae, and Pseudomonas spp. and N. meningitidis. Inan embodiment, the Staphylococcus spp. is S. aureus. In anotherembodiment, the Pseudomonas spp. is Pseudomonas aeruginosa (P.aeruginosa). In another embodiment, the N. meningitidis is N.meningitidis serotype B (MenB). In an embodiment, the sepsis-inducingbacteria are not GBS.

The amino acid sequences of GAPDH from GBS, E. coli, S. aureus, S.pneumoniae, K. pneumoniae, P. aeruginosa and MenB (strain MC58) areidentified herein as SEQ ID NOs: 1-7, respectively.

The amino acid sequence of GAPDH from GBS (UniProt Accession No. Q8E3E8)is identified herein as SEQ ID NO: 1, as follows:—

[SEQ ID NO: 1] MVVKVGINGFGRIGRLAFRRIQNVEGVEVTRINDLTDPNMLAHLLKYDTTQGRFDGTVEVKEGGFEVNGQFVKVSAEREPANTDWATDGVEIVLEATGFFASKEKAEQHIHENGAKKVVITAPGGNDVKTVVFNTNHDILDGTETVISGASCTTNCLAPMAKALQDNFGVKQGLMTTIHAYTGDQMILDGPHRGGDLRRARAGAANIVPNSTGAAKAIGLVIPELNGKLDGAAQRVPVPTGSVTELVATLEKDVTVEEVNAAMKAAANDSYGYTEDPIVSSDIVGISYGSLFDATQTKVQTVDGNQLVKVVSWYDNEMSYTSQLVRTLEYFAKIAK

The amino acid sequence of GAPDH from E. coli (UniProt Accession No.D5D2F1) is identified herein as SEQ ID NO: 2, as follows:—

[SEQ ID NO: 2] MSKVGINGFGRIGRLVLRRLLEVKSNIDVVAINDLTSPKILAYLLKHDSNYGPFPWSVDYTEDSLIVNGKSIAVYAEKEAKNIPWKAKGAEIIVECTGFYTSAEKSQAHLDAGAKKVLISAPAGEMKTIVYNVNDDTLDGNDTIVSVASCTTNCLAPMAKALHDSFGIEVGTMTTIHAYTGTQSLVDGPRGKDLRASRAAAENIIPHTTGAAKAIGLVIPELSGKLKGHAQRVPVKTGSVTELVSILGKKVTAEEVNNALKKATNNNESFGYTDEEIVSSDIIGSHFGSVFDATQTEITAVGDLQLVKTVAWYDNEYGFVTQLIRTLEKFAKL

The amino acid sequence of GAPDH from S. aureus (UniProt Accession No.A6QF81) is identified herein as SEQ ID NO: 3, as follows:—

[SEQ ID NO: 3] MAVKVAINGFGRIGRLAFRRIQEVEGLEVVAVNDLTDDDMLAHLLKYDTMQGRFTGEVEVVDGGFRVNGKEVKSFSEPDASKLPWKDLNIDVVLECTGFYTDKDKAQAHIEAGAKKVLISAPATGDLKTIVFNTNHQELDGSETVVSGASCTTNSLAPVAKVLNDDFGLVEGLMTTIHAYTGDQNTQDAPHRKGDKRRARAAAENIIPNSTGAAKAIGKVIPEIDGKLDGGAQRVPVATGSLTELTVVLEKQDVTVEQVNEAMKNASNESFGYTEDEIVSSDVVGMTYGSLFDATQTRVMSVGDRQLVKVAAWYDNEMSYTAQLVRTLAYLAELSK

Although only the sequence of GAPDH from S. aureus is provided here, allof the available GAPDH sequences from Staphylococcus spp. possess over98% sequence similarity.

The amino acid sequence of GAPDH from S. pneumoniae (UniProt AccessionNo. Q97NL1) is identified herein as SEQ ID NO: 4, as follows:—

[SEQ ID NO: 4] MVVKVGINGFGRIGRLAFRRIQNVEGVEVTRINDLTDPVMLAHLLKYDTTQGRFDGTVEVKEGGFEVNGKFIKVSAERDPEQIDWATDGVEIVLEATGFFAKKEAAEKHLKGGAKKVVITAPGGNDVKTVVFNTNHDVLDGTETVISGASCTTNCLAPMAKALQDNFGVVEGLMTTIHAYTGDQMILDGPHRGGDLRRARAGAANIVPNSTGAAKAIGLVIPELNGKLDGSAQRVPTPTGSVTELVAVLEKNVTVDEVNAAMKAASNESYGYTEDPIVSSDIVGMSYGSLFDATQTKVLDVDGKQLVKVVSWYDNEMSYTAQLVRTLEYFAKIAK

The amino acid sequence of GAPDH from K. pneumoniae (UniProt AccessionNo. B5XRG0) is identified herein as SEQ ID NO: 5, as follows:—

[SEQ ID NO: 5] MSKLGINGFGRIGRLVLRRLLEVDSSLEVVAINDLTSPKVLAYLLKHDSNYGPFPWSVDFTEDALIVNGKTITVYAEKEAQHIPWQAAGAEVIVECTGFYTSAEKSQAHIQAGARKVLISAPAGEMKTIVYNVNDDTLTPDDTIISVASCTTNCLAPMAKVLQDAFGITVGTMTTIHAYTGTQSLVDGPRGKDLRASRAAAENVIPHTTGAAKAIGLVIPALSGKLKGHAQRVPTKTGSVTELVSVLEKKVTADEVNQAMKQAAEGNESFGYTEEEIVSSDIIGSHFGSIYDATQLEIVEAGGVQLVKTVAWYDNEYGFVTQLIRVLEKFAR

The amino acid sequence of GAPDH from P. aeruginosa (UniProt AccessionNo. P27726) is identified herein as SEQ ID NO: 6, as follows:—

[SEQ ID NO: 6] MTIRLAINGFGRIGRNVLRALYTGHYREQLQVVAINDLGDAAVNAHLFQYDSVHGHFPGEVEHDAESLRVMGDRIAVSAIRNPAELPWKSLGVDIVLECTGLFTSRDKAAAHLQAGAGKVLISAPGKDVEATVVYGVNHEVLRASHRIVSNASCTTNCLAPVAQVLHRELGIEHGLMTTIHAYTNDQNLSDVYHPDLYRARSATQSMIPTKTGAAEAVGLVLPELAGKLTGLAVRVPVINVSLVDLTVQVARDTSVDEVNRLLREASEGSPVLGYNTQPLVSVDFNHDPRSSIFDANHTKVSGRLVKAMAWYDNEWGFSNRMLDSALALAAARD

Although only the sequence of GAPDH from P. aeruginosa is provided here,all of the available GAPDH sequences from Pseudomonas spp. possess over98% sequence similarity.

The amino acid sequence of GAPDH from MenB (strain MC58) (UniProtAccession No. Q9JX95) is identified herein as SEQ ID NO: 7, as follows:—

[SEQ ID NO: 7] MSIKVAINGFGRIGRLALRQIEKAHDIEVVAVNDLTPAEMLLHLFKYDSTQGRFQGTAELKDDAIVVNGKEIKVFANPNPEELPWGELGVDVILECTGFFTNKTKAEAHIRAGARKVVISAPGGNDVKTVVYGVNQDILDGSETVISAASCTTNCLAPMAAVLQKEFGVVEGLMTTIHAYTGDQNTLDAPHRKGDLRRARAAALNIVPNSTGAAKAIGLVIPELNGKLDGSAQRVPVASGSLTELVSILERPVTKEEINAAMKAAASESYGYNEDQIVSSDVVGIEYGSLFDATQTRVMTVGGKQLVKTVAWYDNEMSYTCQLVRTLEYFAGKI

Although only the sequence of GAPDH from MenB (strain MC58) is providedhere, all of the available GAPDH sequences from the different serotypesof N. meningitidis possess 97.668% sequence similarity(http://www.uniprot.org/align/A20150610146R80D4XR).

The biochemical characterisation, enzymatic activity and surfacelocalisation of GAPDH protein has been described for GBS [33].Extracellular localisation has also been described for GAPDH from E.coli [34-36], S. aureus [37-39], and S. pneumoniae [40]. The inventorsbelieve that they are the first authors to indicate an extracellularpresence of P. aeruginosa and N. meningitidis GAPDH.

GAPDH is a phylogenetically conserved protein associated with energymetabolism, which is present in every cell type. Microbial GAPDH hasapproximately 30-40% sequence identity with human GAPDH. The amino acidsequence of human GAPDH (UniProt Accession No. P04406) is identifiedherein as SEQ ID NO: 8, as follows:—

[SEQ ID NO: 8] MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENGKLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE

Surprisingly, bacterial GAPDHs share up to 60% of their amino acidsequences, and the inventors have now demonstrated that, even moresurprisingly, the GAPDHs from the preferred sepsis-inducing bacteriadescribed herein (i.e. GBS, E. coli, Staphylococcus spp., S. pneumoniae,K. pneumoniae, Pseudomonas spp. and N. meningitidis) have a particularlyhigh degree of sequence identity (see Example 7). Table 1 below providesthe multiple alignment and sequence similarity percentages that wereobtained from the ClustalW2 server, after submitting the amino acidsequences identified herein as SEQ ID NOs: 1-7 (according to the FASTAformat of the previously indicated UniProt accession numbers). Thesequence alignments are also shown in FIG. 1.

TABLE 1 Sequence comparison of bacterial GAPDH (GBS vs other bacteria)GAPDH sequence comparison Sequence similarity (%) GBS - E. coli 60.61GBS - Staphylococcus spp. 69.05 GBS - S. pneumoniae 91.94 GBS - K.pneumoniae 58.13 GBS - Pseudomonas spp. 44.31 GBS - N. meningitidis(MenB) 70.66

Accordingly, in a preferred embodiment, a peptide of the first aspecthas at least 95%, at least 98%, at least 99% or 100% amino acid sequenceidentity with a peptide found within GAPDH of one or moresepsis-inducing bacteria.

Thus, preferably the peptide has at least 90%, at least 95%, at least98%, at least 99% or even 100% amino acid sequence identity with apeptide found within one or more of the GAPDH sequences identified asSEQ ID NOs: 1-7.

For example, in one preferred embodiment, a peptide of the first aspectmay have at least 90% amino acid sequence identity with a peptide foundin SEQ ID NO: 1 (i.e. GAPDH from GBS). In another preferred embodiment,a peptide of the first aspect may have at least 95% amino acid sequenceidentity with a peptide found in SEQ ID NO: 2 (i.e. GAPDH from E. coli).In yet another preferred embodiment, the peptide may have at least 98%amino acid sequence identity with a peptide found in SEQ ID NO: 4 (i.e.GAPDH from S. pneumoniae). In another preferred embodiment, the peptidemay have at least 95% amino acid sequence identity with a peptide foundin SEQ ID NO: 1 (i.e. GAPDH from GBS), at least 98% amino acid sequenceidentity with a peptide found in SEQ ID NO: 4 (i.e. GAPDH from S.pneumoniae), and at least 99% amino acid sequence identity with apeptide found in SEQ ID NO: 6 (i.e. GAPDH from Pseudomonas spp.), and soon. It will be appreciated that any combination of possible sequenceidentities and possible sepsis-inducing bacteria described herein areenvisaged and constitute part of the invention.

Having surprisingly discovered that GAPDH is highly conserved amongstsepsis-inducing bacteria, the inventors have identified a number ofpeptide sequences that are common to the bacterial GAPDHs, but areabsent from human GAPDH (the expression “common to” can includeconsensus amino acid sequences that are borne out of the alignedbacterial sequences). Four preferred examples of these common peptidesequences are identified herein as SEQ ID NOs: 9-12. The peptides havingthe amino acid sequences of SEQ ID NOs: 9-12 are referred to herein as“Peptides 1-4”, respectively, and these are shown below and in Table 2.

The amino acid sequence of Peptide 1 (i.e. common sequence 1) derivedfrom GAPDH is identified herein as SEQ ID NO: 9, as follows:—

[SEQ ID NO: 9] RIQEVEGLEVTR

The amino acid sequence of Peptide 2 (i.e. common sequence 2) derivedfrom GAPDH is identified herein as SEQ ID NO: 10, as follows:—

[SEQ ID NO: 10] DVTVEENAAM

The amino acid sequence of Peptide 3 (i.e. common sequence 3) derivedfrom GAPDH is identified herein as SEQ ID NO: 11, as follows:—

[SEQ ID NO: 11] EVKDGHLIVNGKV

The amino acid sequence of Peptide 4 (i.e. common sequence 4) derivedfrom GAPDH is identified herein as SEQ ID NO: 12, as follows:—

[SEQ ID NO: 12] EHDAESLRVMGDR

TABLE 2 Peptides derived from bacterial GAPDH for use as a vaccinePeptide Amino acid sequence SEQ ID NO. 1 RIQEVEGLEVTR  9 2 DVTVEENAAM 103 EVKDGHLIVNGKV 11 4 EHDAESLRVMGDR 12

As an illustration, the derivation of Peptides 1 and 2 from the nativesequences found in GADPH from GBS, S. aureus and S. pneumoniae, and thefact that the same sequences do not exist in GAPDH in humans, is shownin FIGS. 2 and 3. Peptides 3 and 4 were derived from the nativesequences found in GADPH from E. coli, K pneumoniae and/or P.aeruginosa, also as identified in FIGS. 2 and/or 3.

The amino acid sequence of each peptide according to the invention maythus be the same as that found in sepsis-inducing bacteria, or it mayvary. However, if it varies, preferably the peptide has an amino acidsequence that is effectively a consensus sequence for thesepsis-inducing bacteria GAPDH, but not for human GADPH.

Accordingly, in one preferred embodiment, a peptide of the first aspecthas an amino acid sequence substantially as set out in any one of SEQ IDNOs: 9-12.

It is expected that GAPDH from the different sepsis-inducing bacteriaharbour further consensus sequences, in addition to those identified asSEQ ID NOs: 9-12, that can be used to generate a peptide according tothe present invention, for use as a vaccine. The peptides may have anysequence, but the sequence must not be substantively shared by GAPDH inhumans.

Alternatively, a native amino acid sequence of GAPDH fromsepsis-inducing bacteria can be used to generate a peptide of theinvention. Table 3 shows putative peptide sequences taken from GAPDH ofthe indicated bacteria, which can be used in a vaccine of the inventionto target at least the indicated bacteria. The peptides have beenidentified as bacterial GAPDH peptides that share no common sequencesimilarity with the human GAPDH isoform, after aligning each bacterialGAPDH amino acid sequence individually with human GAPDH (ClustalW2server, followed by visual inspection and selection). These peptides areillustrative of many more GAPDH peptides that may be found amongst thesurface peptides of the bacteria, and used to create a vaccine of theinvention. Any peptide sequence within the native sequence may be used,but the sequence must not be substantively shared by GAPDH in humans.

TABLE 3 Putative GAPDH peptide sequences derived fromsepsis-inducing bacteria, for use in a vaccine BacteriaAmino acid sequence SEQ ID NO. GBS AFRRIQNVEGVEVTR 13 EVKEGGFEVNGQFVKVSA14 TQTKVQTVDGNQLVK 15 HRGGDLRRARAGAA 16 VEEVNAAMKAAANDSY 17SQLVRTLEYFAKIAK 18 E. coli LRRLLEVKSNIDVV 19 PWSVDYTEDSLIVN 20AGEMKTIVYNVNDDTL 21 GKKVTAEEVNNALK 22 TNNNESFGYTDEEI 23TQTEITAVGDLQLVKTVA 24 YGFVTQLIRTLEKFAKL 25 S. aureus LTDDDMLAHLLKYDTM 26EVVDGGFRVNGKEVKS 27 ATGDLKTIVFNTN 28 HRKGDKRRARAAA 29QDVTVEQVNEAMKNASNESF 30 VEQVNEAMKNASNESF 31 TQTRVMSVGDRQLVKVAA 32SYTAQLVRTLAYLAELSK 33 S. pneumoniae AFRRIQNVEGVEVTR 34 DLTDPVMLAHLLKY 35EVKEGGFEVNGKFIKVSA 36 GGNDVKTVVFNTNHDVL 37 PHRGGDLRRARAGAA 38NVTVDEVNAAMKAASNESY 39 TQTKVLDVDGKQL 40 MSYTAQLVRTLEYFAKIAK 41K. pneumoniae LRRLLEVDSSLEV 42 DLTSPKVLAYLLKH 43 PFPWSVDFTEDALIV 44TVYAEKEAQHIPWQA 45 AGEMKTIVYNVNDDTLTPDDT 46 VSVLEKKVTADEVNQAM 47IIGSHFGSIYDATQ 48 LEIVEAGGVQLVKTVA 49 YGFVTQLIRVLEKFAR 50Pseudomonas spp LRALYTGHYREQLQV 51 DLGDAAVNAHLFQ 52GEVEHDAESLRVMGDRIAVSAI 53 SAIRNPAELPWKSLGVDI 54 VAQVLHRELGIEH 55TIHAYTNDQNLSDVYHPD 56 VYHPDLYRARSATQSMIPTK 57 VQVARDTSVDEVNRLLRE 58GSPVLGYNTQPLVSV 59 ANHTKVSGRLVKAMA 6o MLDSALALAAARD 61 TGHYREQLQ 62 MenBALRQIEKAHDIEV 63 DLTPAEMLLHLFK 64 ELKDDAIVVNGKE 65 HRKGDLRRARAAAL 66NAAMKAAASESYG 67 TQTRVMTVGGKQL 68 TCQLVRTLEYFAGKI 69

Hence, in another preferred embodiment, a peptide of the first aspecthas an amino acid sequence substantially as set out in any one of SEQ IDNOs: 13-69.

Suitably, a peptide of the first aspect comprises 150 amino acids, orless. For example, the peptide preferably comprises less than 100 aminoacids and more preferably less than 50 amino acids. Even more preferablythe peptide comprises less than 30 amino acids and most preferably lessthan 20 amino acids. Suitably, a peptide of the invention comprises atleast 3 amino acids. Preferably, a peptide of the invention comprises atleast 5 amino acids, more preferably at least 8 amino acids, and evenmore preferably at least 10 amino acids. The peptides of the inventioncan be of any length within the above ranges, but they will typically be5-100 amino acids in length, preferably will be 5-50 amino acids inlength and most preferably will be 10-20 amino acids in length.

Suitably, a peptide of the invention should be located at the surface ofeach bacterial GAPDH and present a 3D structure (conformation) similarto the one they possess within the whole protein.

The peptides of the invention can be obtained by any means known in theart, including through recombinant means. For example, the production ofrGAPDH (the whole protein) from GBS has been previously described [41].Desired peptides can be produced in a similar manner. The recombinantproduction of GAPDH (whole protein or peptides) in bacteria other thanGBS is also contemplated as part of the invention. Alternatively, thepeptides can be obtained by protein truncation or synthesised de novo,using techniques well known in the art (such as solid-phase orliquid-phase synthesis). The invention thus extends to nucleic moleculeswhich encode the peptides of the invention.

Hence, according to a second aspect of the invention, there is providedan isolated nucleic acid encoding a peptide according to the firstaspect, or a functional fragment or functional variant thereof.

An experienced investigator in the field would readily be able toidentify suitable nucleic acid sequences that encode a peptide accordingto the first aspect, or a functional fragment or functional variantthereof. The skilled person would hence be readily able to execute thisaspect of the invention, based upon the existing knowledge in the artand/or relevant technical details provided in the published literature(see, for example, [25], which describes a useful method forconstructing and purifying recombinant GAPH).

In an embodiment, the isolated nucleic acid is recombinant or synthetic.In an embodiment, the isolated nucleic acid is a cDNA molecule encodinga peptide according to the first aspect, or a functional fragment orfunctional variant thereof. In an embodiment, the isolated nucleic acidis chemically modified, for example, via the inclusion of a knownmodified nucleotide. In an embodiment, the isolated nucleic acid isoperably linked to a heterologous promoter. In an embodiment, theisolated nucleic acid is bound to a substrate or label or such like.Such modifications are usual in the art and will be known to the skilledperson.

In a third aspect, there is provided a genetic construct comprising anucleic acid according to the second aspect.

Genetic constructs of the invention may be in the form of an expressioncassette, which may be suitable for expression of the encoded peptide ina host cell. The genetic construct may be introduced into a host cellwithout it being incorporated in a vector. For instance, the geneticconstruct, which may be a nucleic acid molecule, may be incorporatedwithin a liposome or a virus particle. Alternatively, a purified nucleicacid molecule (for example, histone-free DNA or naked DNA) may beinserted directly into a host cell by suitable means, for example,direct endocytotic uptake. The genetic construct may be introduceddirectly into cells of a host subject (for example, a bacterial cell) bytransfection, infection, electroporation, microinjection, cell fusion,protoplast fusion or ballistic bombardment. Alternatively, geneticconstructs of the invention may be introduced directly into a host cellusing a particle gun. Alternatively, the genetic construct may beharboured within a recombinant vector, for expression in a suitable hostcell.

Hence, in a fourth aspect of the invention, there is provided arecombinant vector comprising a genetic construct according to the thirdaspect.

The recombinant vector may be a plasmid, cosmid or phage. Suchrecombinant vectors are useful for transforming host cells with thegenetic construct of the fifth aspect, and for replicating theexpression cassette therein. The skilled technician will appreciate thatgenetic constructs of the invention may be combined with many types ofbackbone vector for expression purposes. Recombinant vectors may includea variety of other functional elements including a suitable promoter toinitiate gene expression. For instance, the recombinant vector may bedesigned such that it autonomously replicates in the cytosol of the hostcell. In this case, elements which induce or regulate DNA replicationmay be required in the recombinant vector. Alternatively, therecombinant vector may be designed such that it integrates into thegenome of a host cell. DNA sequences which favour targeted integration(for example, by homologous recombination) may be used.

The recombinant vector may also comprise DNA coding for a gene that maybe used as a selectable marker in the cloning process, i.e. to enableselection of cells that have been transfected or transformed, and toenable the selection of cells harbouring vectors incorporatingheterologous DNA. For example, chloramphenicol resistance is envisaged.Alternatively, the selectable marker gene may be in a different vectorto be used simultaneously with vector containing the gene of interest.The vector may also comprise DNA involved with regulating expression ofthe coding sequence, or for targeting the expressed peptide to a certainpart of the host cell.

Accordingly, in an fifth aspect, there is provided a host cellcomprising a genetic construct according to the third aspect, or arecombinant vector according to the fourth aspect.

The host cell may be a bacterial cell, for example E. coli.Alternatively, the host cell may be an animal cell, for example a mouseor rat cell. It is preferred that the host cell is not a human cell. Thehost cell may be transformed with genetic constructs or vectorsaccording to the invention, using known techniques. Suitable means forintroducing the genetic construct into the host cell will depend on thetype of cell.

In a sixth aspect, there is provided a transgenic host organismcomprising at least one host cell according to the fifth aspect.

The genome of the host cell or the transgenic host organism of theinvention may comprise a nucleic acid sequence encoding a peptide,variant or fragment according to the first aspect. The host organism maybe a multicellular organism, which is preferably non-human. For example,the host organism may be a mouse or rat. The host may be a bacterium.The host may be used for development of a vaccine for immunising asubject against infections with sepsis-inducing bacteria, such as GBS,E. coli, Staphylococcus spp., S. pneumoniae, K. pneumoniae, N.meningitidis and/or Pseudomonas spp. Indeed, knowledge of the amino acidsequences of GAPDH from the different sepsis-inducing bacteria, asdescribed herein, can be harnessed in the development of a vaccine.

As described herein, the inventors were surprised to find that GAPDH isan extracellular virulence factor of the most harmful bacteriaassociated with neonatal sepsis and also sepsis in the elderly anddiabetics. The inventors have surprisingly revealed that bacterial GAPDHinduces, in the host, a production of IL-10 very early upon infection,and that these pathogens are using GAPDH secretion as a form of escapefrom the host immune system (see Examples 5 and 6).

The inventors have also surprisingly discovered that GAPDH acts onimmune cells through interaction with Toll-like receptor 2 (TLR2).Interestingly, the inventors have found that bacterial GAPDH is able toengage TLR2 on the surface of B1 lymphocytes and induce IL-10 productionby these cells (see Examples 1 and 2). The inventors have discoveredthat B1 cells are the major producers of IL-10 upon GAPDH stimulus.

While the inventors were aware from their previous work thatTLR2-mediated IL-10 production plays a key role in the pathophysiologyof neonatal sepsis caused by GBS [42], it was surprising that thisactivity is triggered by GAPDH. According to the literature [43,44], itwas expected that TLR2 recognise bacterial-associated lipoproteins. Theinventors had thus always assumed that TLR2-mediated IL-10 productionwas associated with a GBS lipoprotein. Hence, it was surprising todiscover that bacterial GAPDH could also bind to TLR2 and be responsiblefor the signaling cascade that induces IL-10 production. Given theaforementioned surprising finding that GAPDH is a shared virulencefactor, it was also completely surprising that this activity is sharedby sepsis-inducing bacteria other than GBS (see Example 3).

Other leukocytes, like macrophages, have been found to also produceIL-10 upon bacterial GAPDH recognition, although to a lesser extent thanB1 cells. In addition, the inventors have surprisingly discovered thatGAPDH-induced (i.e. TLR2-mediated) IL-10 production by B1 cells issignificantly augmented in the presence of type I interferon produced bydendritic cells or macrophages, which occurs after recognition ofbacterial antigens by these cells (see Example 4). This represents acompletely novel virulence mechanism, where bacterial structuralantigens and a secreted product act synergistically to induce hostimmunosuppression.

The inventors have also discovered that the tendency of newborns toproduce elevated amounts of IL-10 in the presence of bacterial GAPDH isthe reason for their increased susceptibility to these infections;whilst this was known for GBS [24], it was not known or expected forother sepsis-inducing bacteria (see Example 5). Immunocompromised adultssuch as the elderly and diabetics are also protected from sepsis by theneutralisation of bacterial GAPDH, meaning that the same mechanismobserved in the neonates is true for these groups too (see Examples 12and 13). Moreover, the inventors have found that bacterial GAPDH is alsoa potent inducer of IL-10 in human cord-blood and adult leukocytes,proving that the same mechanism observed in mice is translated intohuman individuals (see Example 6).

As the inventors have studied the main sepsis-inducing bacteria in theirwork, they hypothesise that the same results would be observed in anyother sepsis-inducing bacteria. Taking this into account, the inventorshave developed a GAPDH-based vaccine to prevent infectious diseasescaused by sepsis-inducing bacteria in immunocompromised hosts such asneonates, babies, children, women of fertile age, pregnant women,foetuses, the elderly and diabetics, in particular.

Therefore, according to a seventh aspect of the invention, there isprovided the use of a peptide, fragment or variant according to thefirst aspect, in the development of a vaccine for preventing aninfection with sepsis-inducing bacteria.

The sepsis-inducing bacteria may preferably be selected from a group ofbacteria consisting of GBS, E. coli, Staphylococcus spp., S. pneumoniae,K pneumoniae, Pseudomonas spp. and N. meningitidis. In an embodiment,the Staphylococcus spp. is S. aureus. In another embodiment, thePseudomonas spp. is P. aeruginosa. In another embodiment, the N.meningitidis is MenB. In an embodiment, the sepsis-inducing bacteria arenot GBS.

In an eighth aspect, there is provided a vaccine comprising a peptide,fragment or variant according to the first aspect.

Since GAPDH is a protein which is also present in humans, a vaccineconstituted with the whole bacterial GAPDH protein could raiseautoimmune pathologies. Advantageously, and as exemplified in Examples8-10, when a peptide of the first aspect is used as a vaccine, itresults in a strong antibody response to GAPDH from any of thesepsis-inducing bacteria described here, while concomitantly avoidingautoimmune pathologies. In fact, the use of such a peptide is believedto increase the specificity of the vaccine against each of the bacterialGAPDHs, which, in turn, increases the protection afforded against eachof the bacteria. As described above (see the sequence comparison inTable 1, in some cases, the degree of sequence similarity between GAPDHsfrom different bacteria is not that high. By using different peptides, aspecific immune response towards any of the bacterial GAPDHs can hencebe assured. This would not be possible using a single GAPDH (wholeprotein) as a vaccine. Surprisingly, therefore, the inventors have founda way in which a GAPDH-derived vaccine can be administered to subjectsin need thereof, without causing autoimmune pathologies. With respect toneonates in particular, neonatal B1 cells represent approximately 30% oftotal spleen cells in neonates. On the other hand, adult B1 cellscorrespond to 1-5% of total spleen cells [45]. This is believed by theinventors to reinforce the role of bacterial GAPDH in neonatalsusceptibility to sepsis.

A vaccine of the eighth aspect (or as developed in the seventh aspect)may comprise any of the different peptides described or envisagedherein, or fragments or variants thereof, in any combination and in anynumber. In a preferred embodiment therefore, the vaccine may comprisejust one type of peptide described herein (for example, SEQ ID NO: 9).In another embodiment, the vaccine may comprise any two (for example,SEQ ID NOs: 9 and 10), three (for example, SEQ ID NOs: 9, 10 and 11),four, five, six, seven, eight, nine, ten or more of the peptides, orfragments or variants thereof, described or envisaged herein, and so on.Any combination of the different peptides, or fragments or variantsthereof, are envisaged and form part of the invention.

In one preferred embodiment, the vaccine comprises one or more of thepeptides shown in Table 2 (i.e. having SEQ ID NOs: 9-12), or fragmentsor variants thereof. The vaccine may comprise any one peptide, any twopeptides, any three peptides or indeed all four of the peptides, orfragments or variants thereof.

In another preferred embodiment, the vaccine comprises one or more ofthe peptides shown in Table 3 (i.e. having SEQ ID NOs: 13-69), orfragments or variants thereof. Hence, the vaccine may comprise any oneof the peptides, or any two, three, four, five, six, seven, eight, nine,ten or more of the peptides, or fragments or variants thereof.

In yet another preferred embodiment, the vaccine comprises one or moreof the peptides shown in Table 2 (i.e. having SEQ ID NOs: 9-12) and oneor more of the peptides shown in Table 3 (i.e. having SEQ ID NOs:13-69), or fragments or variants thereof. The vaccine may comprise anyone of the peptides, or any two, three, four, five, six, seven, eight,nine, ten or more of the peptides, or fragments or variants thereof.

In a most preferred embodiment, the vaccine contains all four of thepeptides shown in Table 2 (i.e. having SEQ ID NOs: 9-12), or fragmentsor variants thereof. Purely for convenience, and not to be construed aslimiting in any way, the vaccine containing the combination of thesefour peptides will hereinafter be referred to as Neonatal Vaccine.Although its name refers to neonates in particular, Neonatal Vaccine isintended for use in any of the patient populations described herein andagainst any of the diseases, disorders or conditions described herein.In this regard, pre-term births and stillbirths can be caused by anexacerbated inflammatory response induced by bacterial infections. Inthe cases of bacterial-induced pre-term births and stillbirths, the mostcommon agents are GBS, E. coli and K. pneumoniae, i.e. thesepsis-inducing bacteria described herein.

In a vaccine as described herein, the peptides, fragments or variantsthereof may be linked together to form a larger peptide (or smallprotein). In one embodiment, two or more different peptides (orfragments or variants) are linked together. In another embodiment, twoor more copies of the same peptide (or fragment or variant) are linkedtogether. Linkage may be direct (i.e. having no amino acids inbetweenthe peptides, fragments or variants being linked) or indirect (i.e.having one or more amino acids between the peptides, fragments orvariants being linked, so serving as a ‘spacer’). A pattern of one ormore of the described peptides (or fragments or variants) may berepeated to form the larger peptide/small protein. The repetitions maybe directly adjacent to each other, in a so-called tandem repeat, orthey may be spaced apart by one or more amino acids in each case.Alternatively the linked peptides, fragments or variants may appear in arandom order. Any and all combinations of the above arrangements arealso envisaged and form part of the invention. For example, a largerpeptide may be formed by linking two or more copies of the same peptide,fragment or variant and two or more different peptides, fragments orvariants together, in a pattern, a random order or in a combination ofboth.

Thus, in a preferred embodiment, the vaccine may comprise just one typeof peptide, fragment or variant described herein (for example, SEQ IDNO: 9), but in two or more linked copies of that type. In anotherembodiment, the vaccine may comprise any two (for example, SEQ ID NOs: 9and 10), three (for example, SEQ ID NOs: 9, 10 and 11), four, five, six,seven, eight, nine, ten or more linked copies of the described peptides,fragments or variants, in any of the arrangements described immediatelyabove.

In one preferred embodiment, the vaccine comprises one or more linkedcopies of the peptides shown in Table 2 (i.e. having SEQ ID NOs: 9-12),or fragments or variants thereof. The vaccine may comprise any onepeptide, any two peptides, any three peptides or indeed all four of thepeptides, or fragments or variants thereof, in any linkage arrangementas described herein.

In another preferred embodiment, the vaccine comprises one or morelinked copies of the peptides shown in Table 3 (i.e. having SEQ ID NOs:13-69), or fragments or variants thereof. Hence, the vaccine maycomprise any one of the peptides, or any two, three, four, five, six,seven, eight, nine, ten or more of the peptides, or fragments orvariants thereof, in any linkage arrangement as described herein.

In yet another preferred embodiment, the vaccine comprises one or moreof the peptides shown in Table 2 (i.e. having SEQ ID NOs: 9-12) and oneor more of the peptides shown in Table 3 (i.e. having SEQ ID NOs:13-69), or fragments or variants thereof, in any linkage arrangement asdescribed herein. The vaccine may comprise any one of the peptides, orany two, three, four, five, six, seven, eight, nine, ten or more of thepeptides, or fragments or variants thereof, in any linkage arrangementas described herein.

In a most preferred embodiment, the vaccine is Neonatal Vaccine, i.e.containing all four of the peptides shown in Table 2 (i.e. having SEQ IDNOs: 9-12), or fragments or variants thereof, in any linkage arrangementas described herein.

Thus, a vaccine of the eighth aspect (or as developed in the seventhaspect) may comprise any two, three, four, five, six, seven, eight,nine, ten or more peptides, fragments or variants of the first aspect,wherein at least two of the peptides, fragments and/or variants arelinked together. Any two, three, four, five, six, seven, eight, nine,ten or more of the peptides, fragments or variants may be linkedtogether in this respect.

The inventors were surprised to observe that the peptides derived fromGAPDH of sepsis-inducing bacteria, and vaccines comprising thesepeptides, are able to elicit a protective antibody response. Inparticular, the antibodies raised against the peptides are able tospecifically recognise, and neutralise, GAPDH of sepsis-inducingbacteria.

Accordingly, in a ninth aspect, there is provided the use of a peptide,fragment or variant according to the first aspect, or a vaccineaccording to the eighth aspect, for stimulating an immune response.

Preferably, the immune response includes the production of antibodiesthat are specific to GAPDH of one or more species of sepsis-inducingbacteria. The sepsis-inducing bacteria may be GBS, E. coli,Staphylococcus spp., S. pneumoniae, K pneumoniae, N. meningitidis and/orPseudomonas spp. In an embodiment, the Staphylococcus spp. is S. aureus.In another embodiment, the Pseudomonas spp. is P. aeruginosa. In anotherembodiment, the N. meningitidis is MenB. Thus, the GAPDHs for which theantibodies have specificity may be those having the amino acid sequencesprovided as SEQ ID NOs: 1-7. In an embodiment, the sepsis-inducingbacteria are not GBS.

As GAPDH is a ubiquitous protein and, as demonstrated herein, conservedamongst the sepsis-inducing bacteria, the peptides, fragments andvariants of the invention are able to induce protection against all ofthe different serotypes of sepsis-inducing bacteria, which isadvantageous.

Previously, glycoconjugate vaccines against nine GBS serotypes have beenshown to be immunogenic in animals, but the existence of distinctepitope-specific capsular serotypes prevented the development of aglobal GBS vaccine [46,47]. In contrast, GAPDH is structurally conservedin all eight published GBS genomes (identity >99.8%). As the inventorshave previously described, anti-GAPDH immunoglobulin G (IgG) antibodiespurified from sera of GAPDH-immunised mice or rabbits have thus beenused to demonstrate the presence of GAPDH in culture supernatants of 10unrelated GBS clinical isolates belonging to different serotypes and/orMLSTypes [24]. As GBS GAPDH displays only 44.7, 45.8 and 44.0% aminoacid identity with rabbit, mice and human GAPDH, respectively, however,the previously described ‘GBS vaccine’ does not induce any autoimmunityupon administration in mammals [24]. The same is true for the peptides,fragments and variants described herein, given the inventors' presentdiscovery that GAPDH is conserved between all sepsis-inducing bacteria.

The use of the peptide, fragment or variant may be an in vitro, in vivoor ex vivo use.

Preferably, the use of the peptide, fragment or variant thereof is an invitro or ex vivo use for the production of antibodies. In a particularlypreferred embodiment, the in vitro or ex vivo use is for the productionof monoclonal or polyclonal antibodies.

Such uses may involve the interaction of a peptide, fragment or variantof the first aspect with antibody-producing cells in vitro or ex vivo,such that antibodies that are specific for GAPDH of one or more speciesof sepsis-inducing bacteria may be produced. Suitable antibody-producingcells and techniques for contacting the same with the peptides,fragments or variants of the invention in order to produce antibodiesare described in the art and will be known to the skilled person. Forexample, blood products of immune people and/or non-human immune animalsmay be used to generate the antibodies. Alternatively, the peptides,fragments or variants of the first aspect may be used to producehybridomas specific for different epitopes of bacterial GAPDH. Standardtechniques available in the art could be used to produce the hybridoma.

In another preferred embodiment, the use of the peptide, fragment orvariant thereof is an in vivo use, i.e. for stimulating an immuneresponse in a subject.

The peptide, fragment or variant may be administered directly into asubject to be vaccinated on its own, i.e. just one or more isolatedpeptides having at least 90% amino acid sequence identity with a peptidefound within GAPDH of one or more sepsis-inducing bacteria, and havingless than 10% amino acid sequence identity with a peptide found withinhuman GAPDH, or a functional fragment or functional variant thereof.

The peptide, fragment or variant may be administered by any means,including by injection or mucosally. Preferably, the peptide, fragmentor variant is administered intra-muscularly, sub-cutaneously,intra-venously or intra-dermically. It will be appreciated thatadministration, into a subject to be vaccinated, of a peptide, fragmentor variant of the invention will result in the production ofcorresponding antibodies exhibiting immunospecificity for the peptide,fragment or variant, and that these antibodies aide in ameliorating ortreating an existing infection, and preventing a subsequent infection,with sepsis-inducing bacteria.

In a preferred embodiment, therefore, the peptides, fragments orvariants thereof are for stimulating the production of antibodies thatare specific to GAPDH of sepsis-inducing bacteria, such as GBS, E. coli,Staphylococcus spp., S. pneumoniae, K. pneumoniae, N. meningitidisand/or Pseudomonas spp.

The skilled person will appreciate that there are various ways in whicha vaccine could be made based on the antigenic peptides, fragments andvariants described herein, such as the peptides represented as SEQ IDNOs: 9-69, and fragments and variants thereof. For example, geneticallyengineered vaccines may be constructed where the heterologous antigen(i.e. the peptide, fragment or variant thereof) is fused to a promoteror gene that facilitates expression in a host vector (for example, abacterium, such as E. coli, or a virus such as Adenovirus).

The vaccine may comprise an excipient, which may act as an adjuvant.Thus, in an embodiment, the antigenic peptides, fragments or variants inthe vaccine may be combined with a microparticulate adjuvant, forexample a liposome or an immune stimulating complex (ISCOM). Thepeptides, fragments or variants may be combined with an adjuvant, suchas cholera toxin, or a squalene-like molecule. Any adjuvant may be used,such as, for example, aluminium hydroxide (alum), tetanus toxoid ordiphtheria toxin. A vehicle may suitably be used for the adjuvant, whichmay include, but is not limited to, water, phosphate buffered saline(PBS), a polyol or a dextrose solution.

The peptides, fragments or variants thereof may also suitably be used inconjunction with a carrier protein, so as to increase the effective sizeof the peptide, fragment or variant. In this manner the immune systemwill not only recognise the peptide or fragment or variant thereof, butwill have memory to it too. The peptides, fragments or variants thereofmay be associated with any carrier protein, such as, for example,hemocyanin from keyhole limpet (KLH).

A vaccine of the invention thus suitably comprises one or more peptidesas described herein, or one or more fragments or variants thereof,together with an adjuvant and/or a carrier protein. Any of the describedpeptides may be used, whether alone or in combination with any of theother described peptides. As described herein, Neonatal Vaccine containsall four of the peptides shown in Table 2 (i.e. having SEQ ID NOs:9-12), or fragments or variants thereof. However, any combination of thedescribed peptides may instead be used. The adjuvant may be any that islicensed for human use, such as alum, tetanus toxoid (TT) or diphtheriatoxin (DT). The carrier protein(s) can be KLH, bovine serum albumin(BSA), ovalbumin (OVA), TT and/or DT. Any other carrier protein suitablefor use in humans may also or alternatively be used in the vaccine.

Example 7 describes one way in which a vaccine may be prepared. Firstly,one or more of the peptides, fragments or variants thereof according tothe first aspect may be chosen as an antigen against which asubsequently vaccinated subject will produce corresponding antibodies.The sequence of the designated gene or nucleic acid molecule encodingthe designated peptide, fragment or variant may then be cloned into asuitable vector to form a genetic construct of the third aspect of theinvention, using techniques known in the art.

The DNA sequence encoding the designated antigen may be inserted intoany known target gene from the host bacterial cell that encodes a knownprotein. The DNA sequence encoding the antigen may be inserted into amultiple cloning site. It will be appreciated that insertion into anygene is permissible as long as the growth and function of the hostorganism is not impaired, i.e. the insertion is functionally redundant.

The thus created genetic construct may be used to transform a vegetativemother cell by double cross-over recombination. Alternatively, thegenetic construct may be an integrative vector, which may be used totransform a vegetative mother cell by single cross-over recombination.

The construct may comprise a drug-resistance gene that is selectable inthe host cell, for example chloramphenicol resistance. Afterconfirmation of the plasmid clone, the plasmid may then be introducedinto a host cell by suitable means. Transformation may be DNA-mediatedtransformation or by electroporation. Selection may be achieved bytesting for drug resistance carried by the plasmid, and now introducedinto the genome.

Expression of the hybrid or chimeric gene may be confirmed using Westernblotting and probing of size-fractionated proteins (sodium dodecylsulphate polyacrylamide gel electrophoresis; SDS-PAGE) using antibodiesthat recognise the introduced antigen (i.e. the peptide, fragment orvariant derived from bacterial GAPDH). If the antigenic gene or nucleicacid fused to the host gene is correctly expressed, a new band appearswhich is recognised only by the antibody, and not normally found in thehost. Other techniques that may be used are immunofluorescencemicroscopy and fluorescence-activated cell sorting (FACS) analysis thatcan show surface expression of antigens on the host's surface.

The resultant vaccines may be administered to a subject by any route,including intramuscular, subcutaneous, intradermic, oral, inhalable,intranasal, rectal and intravenous routes. Oral administration may besuitably via a tablet, a capsule or a liquid suspension or emulsion.Alternatively the vaccines may be administered in the form of a finepowder or aerosol via a Dischaler® or Turbohaler®. Intranasaladministration may suitably be in the form of a fine powder or aerosolnasal spray or modified Dischaler® or Turbohaler®. Rectal administrationmay suitably be via a suppository.

A vaccine of the invention is formulated for administration to anysubject in need thereof, and particularly an immunocompromised host suchas a neonate, baby, child, woman of fertile age, pregnant woman, foetus,an elderly subject or a diabetic. The vaccines need not only beadministered to those already showing signs of an infection, or thoseconsidered to be immunocompromised or at greater risk of an infection,by sepsis-inducing bacteria. Rather, a vaccine can be administered toapparently healthy subjects as a purely preventative measure against thepossibility of such an infection in future. For example, it can beadministered as part of a general vaccination programme toimmunocompromised hosts such as neonates, babies, children, women offertile age, pregnant women, foetuses, the elderly and diabetics.

As used herein, “immunocompromised” means having a compromised immunesystem, as exemplified by neonates, babies, children, women of fertileage, pregnant women, foetuses, the elderly and diabetics.

A vaccine of the invention can be formulated for administration to theindicated subjects at any age. It is intended that it will beadministered to children of any age, including neonates, babies,toddlers and children of school age. It is intended that it will beadministered to pregnant women and women of fertile age, so as toprotect both the mother and foetus from infection. It is also intendedthat it will be administered to elderly subjects at any time duringtheir old age, and to diabetics at any point in their lifetime.

The terms ‘neonate’ and ‘newborn’, as used herein, can refer to a childfrom birth to around one month old. The terms apply to prematureinfants, postmature infants and full term infants. Before birth, theterm ‘foetus’ is used.

The terms ‘baby’ and ‘infant’, as used herein, can refer to youngchildren between the ages of around one month and around one or twoyears of age (i.e. the age when a child learns to walk and talk, whenthe term ‘toddler’ may be used instead).

The term ‘child’, as used herein, refers to young children, coveringthose from toddlers to around 12 years of age, i.e. the pre-teens.

The term ‘elderly’, as used herein, refers to subjects of advanced age.For example, it can refer to men and women aged 60 or over, 65 or over,70 or over, 75 or over, or 80 or over. Non-human subjects in thecorresponding later years of life are also encompassed by this term.

The term ‘diabetic’, as used herein, refers to a person suffering fromdiabetes mellitus type 1 (also known as juvenile or insulin-dependentdiabetes), at any stage of the disease. This is the only type ofdiabetes that is associated with immune system pathology, rendering thepatients immunocompromised.

A vaccine of the invention can be administered simultaneously with otherexisting vaccines, for example, those recommended for immunocompromisedhosts such as babies, children, women of fertile age, pregnant women,the elderly and diabetics (such as, for example, tetanus and diphtheriavaccine).

A vaccine of the invention can be administered to women of fertile ageby intramuscular, subcutaneous, intradermic, oral, intranasal orintra-venous route, in particular. A boost for this vaccine is intendedin the third trimester of gestation. The vaccine is intended to protectwomen of fertile age from peri-natal infections caused bysepsis-inducing bacteria including GBS, E. coli, Staphylococcus spp., S.pneumoniae, K. pneumoniae, Pseudomonas spp. and N. meningitidis. Unborninfants (foetuses) benefit from passive immunity acquired when theirmothers' antibodies cross the placenta to reach the developing child,especially in the third trimester. As illustrated by the Examples, avaccine of the invention can also prevent premature births andstillbirths caused by in utero infections due to the ascending ofbacteria (such as GBS, E. coli and Klebsiella spp.) from the genitaltract into the amniotic fluid.

A suitable dosing regime may be used depending on the organism to bevaccinated. For example, for a human subject to be vaccinated, normallythree doses of 10 mg/kg as a tablet or capsule) at intervals of twomonths may be used. Blood may be withdrawn for analysis of serum (IgG)responses. Saliva, vaginal fluids or faeces may be taken for analysis ofmucosal (secretory IgA) responses. Indirect enzyme-linked immunosorbentassay (ELISA) may be used to analyse antibody responses in serum andmucosal samples, to gauge the efficacy of the vaccination.

As described in the Examples, the inventors have shown that the peptidesof the invention are able to induce a protective antibody responsetoward sepsis-inducing bacteria. The inventors have demonstrated in theExamples that a vaccine of the invention is able to prevent an infectionby sepsis-inducing bacteria. Preferably, the vaccine is for preventing aGBS, E. coli, Staphylococcus spp., S. pneumoniae, K pneumoniae, N.meningitidis and/or Pseudomonas spp. infection. In the development of avaccine, and as described above, it is preferred that any or all of SEQID NOs: 9-69, or fragments or variants thereof, may be used as anantigen for triggering an immune response in a subject which is to bevaccinated. The vaccine is a prophylactic; that is to say, the peptides,fragments or variants described herein may be used to prevent aninfection, including preventing a relapse/recolonisation of a previousinfection.

Therefore, in a tenth aspect, there is provided a peptide, fragment orvariant of the first aspect, for use in therapy.

In an eleventh aspect, the invention provides a peptide, fragment orvariant of the first aspect or a vaccine according to the eighth aspect,for use in preventing an infection by sepsis-inducing bacteria.

Furthermore, according to a twelfth aspect of the invention, there isprovided a method of preventing an infection by sepsis-inducingbacteria, the method comprising administering, to a subject in need ofsuch treatment, a peptide, fragment or variant according to the firstaspect or a vaccine according to the eighth aspect.

A peptide, fragment, variant or vaccine of the invention can preventsystemic infections caused by one or more of at least seven differentpathogens, being the most common causes of sepsis, preferably: GBS, E.coli, Staphylococcus spp., S. pneumoniae, K pneumoniae, Pseudomonas spp.and N. meningitidis. In an embodiment, the Staphylococcus spp. is S.aureus. In another embodiment, the Pseudomonas spp. is P. aeruginosa. Inanother embodiment, the N. meningitidis is MenB. In an embodiment, thesepsis-inducing bacteria are not GBS. A peptide, fragment, variant orvaccine of the invention can thus prevent sepsis or any other disease,disorder or condition caused by an infection of sepsis-inducingbacteria. These other diseases, disorders or conditions includepneumonia, meningitis, endocarditis, enterocolitis, urinary tractinfections, soft tissue infections, gastrointestinal infections,bloodstream infections and encephalitis.

In a preferred embodiment, a peptide, fragment, variant or vaccine ofthe invention is used to prevent a premature birth and/or stillbirth. Asexplained herein, premature births and stillbirths are caused by inutero infections due to the ascending of bacteria (such as GBS, E. coliand Klebsiella spp.) from the genital tract into the amniotic fluid. Byvaccinating the expectant mother with a peptide, fragment, variant orvaccine of the invention, passive immunity with antibodies raisedagainst such an antigen is provided in the unborn offspring. Foetusesand neonates can thus be protected against infection through a maternalvaccination.

The inventors have realised that knowledge of GAPDH being secreted byall of the sepsis-inducing bacteria, and in particular, the sequencesimilarity of these secreted GAPDHs, can also be harnessed in thepreparation of useful therapeutic drugs for treating, preventing orameliorating infections by sepsis-inducing bacteria. For example, anyagent which blocks the binding of the secreted GADPH with a target humanor animal cell can be used as a medicament to prevent, treat orameliorate an infection in that target cell.

The agent which is capable of blocking the binding of the secreted GADPHwith a human or animal cell may be an antibody. For example, an antibodyexhibiting specificity to any of the peptides, fragments or variantsdescribed herein, including those having an amino acid sequence setforth in SEQ ID NOs: 9-69, would be capable of blocking binding of thesecreted GAPDH to a human or animal cell. For example, if the vaccine isadministered mucosally it will generate secretory IgA at the mucosalsurface, and the antibody (sIgA) would block binding of the GAPDH to thehost cell epithelium. If the vaccine is administered systemically itwill induce the production of IgG, which will block the binding ofbacterial GAPDH to TLR2 on the surface of B1 cells and prevent earlyIL-10 production by these cells.

Therefore, according to a thirteenth aspect of the invention, there isprovided an antibody that is specific for GAPDH of one or more speciesof sepsis-inducing bacteria, said antibody being raised against apeptide, fragment or variant of the first aspect.

The term ‘specific for’, as used herein in connection with antibodies,can mean that the variable regions of the antibodies recognise and bindtheir targets (e.g. a peptide or polypeptide) exclusively (i.e. able todistinguish the target peptide or polypeptide from other similarpeptides or polypeptides despite sequence identity, homology orsimilarity found in the family of peptides or polypeptides).

As above, the amino acid sequences of the peptides, fragments orvariants of the first aspect of the invention may be sequences that arefound within a native bacterial GAPDH sequence. Such sequences maytherefore represent target epitopes on the bacterial GAPDH, which can beexploited in the blocking of the binding of the secreted GAPDH to ahuman or animal cell.

Accordingly, in a fourteenth aspect, there is provided an antibody thatis specific for an epitope found in GAPDH of one or more species ofsepsis-inducing bacteria, wherein the epitope has an amino acid sequencesubstantially as set out in any one of SEQ ID NOs: 9-69.

The antibodies of the thirteenth and fourteenth aspects are capable ofblocking the binding of GAPDH secreted by one or more species ofsepsis-inducing bacteria to a human or animal cell. As above, the cellmay be an epithelial cell or a B1 cell. Other leukocytes, such asmacrophages, are also envisaged. For example, the antibody may be sIgAand capable of blocking the binding of the GAPDH to a human or animalepithelial cell. Alternatively the antibody may be IgG and capable ofblocking the binding of the GAPDH to TLR2 on the surface of human oranimal leukocytes, such as B1 cells or macrophages. Such antibodies areconsequently suitable for blocking or neutralising GAPDH-induced IL-10production in the human or animal cells. Both monoclonal and polyclonalantibodies are encompassed by the invention.

According to a fifteenth aspect of the invention, there is provided amethod of producing antibodies that are specific for GAPDH of one ormore species of sepsis-inducing bacteria, the method comprising the stepof contacting antibody-producing cells with a peptide, fragment orvariant of the first aspect, or a vaccine according to the eighthaspect.

Preferably, the sepsis-inducing bacteria referred to in the aboveaspects of the invention are GBS, E. coli, Staphylococcus spp., S.pneumoniae, K pneumoniae, N. meningitidis and/or Pseudomonas spp. In anembodiment, the Staphylococcus spp. is S. aureus. In another embodiment,the Pseudomonas spp. is P. aeruginosa. In another embodiment, the N.meningitidis is MenB. Thus, the GAPDHs for which the antibodies havespecificity, or to which the antibodies bind, may be those having theamino acid sequences identified as SEQ ID NOs: 1-7. In an embodiment,the sepsis-inducing bacteria are not GBS.

The methods of the invention may be in vitro, in vivo or ex vivomethods.

Suitable in vitro and ex vivo methods are those as described in theninth aspect of the invention, i.e. methods for the production of(monoclonal or polyclonal) antibodies that are specific for GAPDH of oneor more species of sepsis-inducing bacteria.

Suitable in vivo methods are those as described in the ninth aspect ofthe invention, i.e. methods of vaccination.

In a sixteenth aspect, there is provided a method of treating,ameliorating or preventing an infection by sepsis-inducing bacteria, themethod comprising administering, to a subject in need of such treatment,an antibody according to the thirteenth or fourteenth aspect or avaccine according to the eighth aspect.

Preferably, the antibody is capable of treating, ameliorating orpreventing an infection with GBS, E. coli, Staphylococcus spp., S.pneumoniae, K pneumoniae, N. meningitidis and/or Pseudomonas spp. In anembodiment, the Staphylococcus spp. is S. aureus. In another embodiment,the Pseudomonas spp. is P. aeruginosa. In another embodiment, the N.meningitidis is MenB. Thus, the GAPDHs for which the antibodies havespecificity are preferably those having the amino acid sequencesprovided as SEQ ID NOs: 1-7. In an embodiment, the sepsis-inducingbacteria are not GBS.

An antibody of the invention can thus prevent, treat or amelioratesepsis or any other disease, disorder or condition caused by aninfection of sepsis-inducing bacteria. These other diseases, disordersor conditions include pneumonia, meningitis, endocarditis,enterocolitis, urinary tract infections, soft tissue infections,gastrointestinal infections, bloodstream infections and encephalitis.Antibodies of the thirteenth and fourteenth aspects of the invention,for use in therapy, and particularly for use in preventing, treating orameliorating an infection by sepsis-inducing bacteria, includingpreventing, treating or ameliorating the aforementioned diseases,disorders and conditions, are therefore also provided.

Preferably, the antibody is raised against a peptide, fragment orvariant as defined in the first aspect of the invention, or a vaccine asdefined in the eighth aspect of the invention.

As discussed above in connection with the twelfth aspect of theinvention, antibodies raised against a peptide, fragment, variant orvaccine of the invention can pass to an unborn baby across the mother'splacenta or in the mother's milk during lactation. Such passiveimmunity, provided in the unborn offspring, can protect againstinfection by sepsis-inducing bacteria and, prevent a premature birthand/or stillbirth. In a preferred embodiment, therefore, the method ofthe sixteenth aspect is a method of preventing infection in an unbornbaby and, thus, a method of preventing a premature birth and/orstillbirth. In this embodiment, the antibody would be administered tothe expectant mother, as a suitable strategy to substitute intrapartumantibiotic prophylaxis. Antibodies of the thirteenth and fourteenthaspects of the invention, for use in preventing, premature birth and/orstillbirth, are therefore also provided.

As used herein, the term ‘antibody’ includes not just whole IgG, butportions thereof, including Fab and F(ab′)2 fragments, too. It alsoincludes sIgA.

Thus, in addition to vaccination using a vaccine of the invention, theantibodies elicited with the peptides, fragments, variants or vaccinesdescribed herein, whether the whole sIgA or IgG antibody or portionsthereof, including Fab or F(ab′)2 fragments, can be used as a treatmentfor infected individuals and/or in mothers that did not receive thevaccine, particularly as follows:

a) A therapeutic approach to be administered in neonates with proven orsuspected sepsis or infection by sepsis-inducing bacteria—whole IgG orFab/F(ab′)2 fragments;b) A preventive approach against infection by sepsis-inducing bacteria,to be administered in neonates born from mothers that did not receivethe vaccine—whole IgG or Fab/F(ab′)2 fragments;c) A preventive approach against infection by sepsis-inducing bacteria,to be administered in mothers in the third trimester of vaccination thatdid not receive the vaccine or in women of fertile age—whole IgG; andd) A therapeutic approach for expectant mothers or women of fertile agewith proven sepsis or invasive infections caused by sepsis-inducingbacteria.

Passive administration of anti-GAPDH antibodies constitutes asignificant improvement over the current therapeutic approaches based onantibiotic administration, which causes the selection of resistantstrains. Passive immunity results when a person is given anothersubject's antibodies. When these antibodies are introduced into theperson's body, the ‘loaned’ antibodies help prevent or fight certaininfectious diseases. The protection offered by passive immunisation isshort-lived, usually lasting only a few weeks or months, but it helpsprotect right away.

As demonstrated herein, passive immunity can be induced artificiallywhen antibodies are given as a medication to a non-immune individual. Asabove, these antibodies may come from the pooled and purified bloodproducts of immune people or from non-human immune animals, such ashorses, sheep and rabbits. As shown in the Examples, passiveadministration of antibodies to newborn mice confers protection fromlethal infection with GBS, E. coli, S. pneumoniae and S. aureus. Theseantibodies are to be administered to mothers who were not vaccinatedwith a vaccine of the invention and/or in newborns from non-vaccinatedmothers. As discussed herein, passive immunity can also be induced infoetuses by administration of the peptides, agents, vaccines, antibodiesand medicaments of the invention to expectant mothers. Unborn infants(foetuses) benefit from passive immunity acquired when their mothers'antibodies cross the placenta to reach the developing child, especiallyin the third trimester. This is therefore a suitable strategy tosubstitute intrapartum antibiotic prophylaxis.

It will be appreciated that peptides, agents, vaccines, antibodies andmedicaments according to the invention may be used in a monotherapy(i.e. the sole use of that peptide, agent, vaccine, antibody ormedicament), for treating, ameliorating or preventing an infection withsepsis-inducing bacteria. Alternatively, peptides, agents, vaccines,antibodies and medicaments according to the invention may be used as anadjunct to, or in combination with, known therapies for treating,ameliorating, or preventing infections with sepsis-inducing bacteria.For example, the peptide, agent, vaccine, antibody or medicament may beused in combination with known agents for treating with sepsis-inducingbacteria infections. For example, the peptide, agent, vaccine, antibodyor medicament may be used in combination with known agents for treatingneonatal sepsis caused by fungi or viruses. It can be used incombination with known anti-retroviral agents.

There is no restriction on which peptide, agent, vaccine, antibody ormedicament as described herein should be administered to which patient.Rather, it is intended that any of the peptides, agents, vaccines,antibodies and medicaments described herein can be administered to anypatient as described herein. It is expressly intended by the inventors,in fact, that each and every combination of peptide, agent, vaccine,antibody or medicament, and indicated patient group, is encompassed bythis invention. The invention thus includes each and every possiblecombination of therapeutic agent and indicated patient group. The use ofNeonatal Vaccine in immunocompromised hosts such as neonates, babies,children, women of fertile age, pregnant women, foetuses, the elderlyand diabetics is preferred.

The peptides, agents, vaccines, antibodies and medicaments according tothe invention may be combined in compositions having a number ofdifferent forms depending, in particular, on the manner in which thecomposition is to be used. Thus, for example, the composition may be inthe form of a powder, tablet, capsule, liquid, ointment, cream, gel,hydrogel, aerosol, spray, micellar solution, transdermal patch, liposomesuspension or any other suitable form that may be administered to aperson or animal in need of treatment. It will be appreciated that thevehicle of medicaments according to the invention should be one which iswell-tolerated by the subject to whom it is given, and preferablyenables delivery of the agents across the blood-brain barrier.

Medicaments comprising peptides, agents, vaccines and antibodies of theinvention may be used in a number of ways. For instance, oraladministration may be required, in which case the agents may becontained within a composition that may, for example, be ingested orallyin the form of a tablet, capsule or liquid. Compositions comprisingpeptides, agents, vaccines, antibodies and medicaments of the inventionmay be administered by inhalation (for example, intranasally).Compositions may also be formulated for topical use. For instance,creams or ointments may be applied to the skin.

Peptides, agents, vaccines, antibodies and medicaments according to theinvention may also be incorporated within a slow- or delayed-releasedevice. Such devices may, for example, be inserted on or under the skin,and the medicament may be released over weeks or even months. The devicemay be located at least adjacent the treatment site. Such devices may beparticularly advantageous when long-term treatment with agents usedaccording to the invention is required and which would normally requirefrequent administration (for example, at least daily injection).

In a preferred embodiment, peptides, agents, vaccines, antibodies andmedicaments according to the invention may be administered to a subjectby injection into the blood stream or directly into a site requiringtreatment. Injections may be intravenous (bolus or infusion) orsubcutaneous (bolus or infusion), or intradermal (bolus or infusion).

It will be appreciated that the amount of the peptide, agent, vaccine,antibody or medicament that is required is determined by its biologicalactivity and bioavailability, which in turn depends on the mode ofadministration, the physiochemical properties of the peptide, agent,vaccine, antibody and medicament, and whether it is being used as amonotherapy or in a combined therapy. The frequency of administrationwill also be influenced by the half-life of the agent within the subjectbeing treated. Optimal dosages to be administered may be determined bythose skilled in the art, and will vary with the particular agent inuse, the strength of the pharmaceutical composition, the mode ofadministration, and the advancement of the bacterial infection.Additional factors depending on the particular subject being treatedwill result in a need to adjust dosages, including subject age, weight,gender, diet and time of administration.

Generally, a daily dose of between 0.001 μg/kg of body weight and 10mg/kg of body weight of peptide, agent, vaccine, antibody or medicamentaccording to the invention may be used for treating, ameliorating, orpreventing bacterial infection, depending upon which peptide, agent,vaccine, antibody or medicament is used. More preferably, the daily doseis between 0.01 μg/kg of body weight and 1 mg/kg of body weight, morepreferably between 0.1 μg/kg and 100 μg/kg body weight, and mostpreferably between approximately 0.1 μg/kg and 10 μg/kg body weight.

The peptide, agent, vaccine, antibody or medicament may be administeredbefore, during or after onset of the bacterial infection. Daily dosesmay be given as a single administration (for example, a single dailyinjection). Alternatively, the peptide, agent, vaccine, antibody ormedicament may require administration twice or more times during a day.As an example, peptides, agents, vaccines, antibodies and medicamentsmay be administered as two (or more depending upon the severity of thebacterial infection being treated) daily doses of between 0.07 μg and700 mg (i.e. assuming a body weight of 70 kg). A patient receivingtreatment may take a first dose upon waking and then a second dose inthe evening (if on a two-dose regime) or at 3- or 4-hourly intervalsthereafter. Alternatively, a slow release device may be used to provideoptimal doses of peptides, agents, vaccines, antibodies and medicamentsaccording to the invention to a patient without the need to administerrepeated doses. Known procedures, such as those conventionally employedby the pharmaceutical industry (for example, in vivo experimentation,clinical trials, etc.), may be used to form specific formulations of thepeptides, agents, vaccines, antibodies and medicaments according to theinvention and precise therapeutic regimes (such as daily doses of theagents and the frequency of administration).

In a seventeenth aspect of the invention, there is provided asepsis-inducing bacteria treatment composition comprising an antibody ofthe thirteenth or fourteenth aspect of the invention, and optionally apharmaceutically acceptable vehicle.

The term “sepsis-inducing bacteria treatment composition” or“anti-sepsis-inducing bacteria composition” can mean a pharmaceuticalformulation used in the therapeutic amelioration, prevention ortreatment of sepsis-inducing bacteria infections in a subject.

The invention also provides in a eighteenth aspect, a process for makingthe composition according to the seventeenth aspect, the processcomprising combining a therapeutically effective amount of an antibodyof the thirteenth or fourteenth aspect of the invention, with apharmaceutically acceptable vehicle.

A “therapeutically effective amount” of an agent (for example, anantibody of the invention) is any amount which, when administered to asubject, is the amount of agent that is needed to treat the infection,or produce the desired effect.

For example, the therapeutically effective amount of agent (for example,antibody) used may be from about 0.001 mg to about 1000 mg, andpreferably from about 0.01 mg to about 500 mg. It is preferred that theamount of agent is an amount from about 0.1 mg to about 100 mg, and mostpreferably from about 0.5 mg to about 50 mg. As a guide, the dose ofantibody used in the neonatal mice in the Examples described herein was40 mg/kg.

A “pharmaceutically acceptable vehicle” as referred to herein, is anyknown compound or combination of known compounds that are known to thoseskilled in the art to be useful in formulating pharmaceuticalcompositions.

A “subject”, as used herein, may be a vertebrate, mammal or domesticanimal. Hence, peptides, agents, vaccines, antibodies and medicamentsaccording to the invention may be used to treat any mammal, for examplelivestock (for example, a horse), pets, or may be used in otherveterinary applications. Most preferably, the subject is a human being.

In one embodiment, the pharmaceutically acceptable vehicle may be asolid, and the composition may be in the form of a powder or tablet. Asolid pharmaceutically acceptable vehicle may include one or moresubstances which may also act as flavouring agents, lubricants,solubilisers, suspending agents, dyes, fillers, glidants, compressionaids, inert binders, sweeteners, preservatives, dyes, coatings ortablet-disintegrating agents. The vehicle may also be an encapsulatingmaterial. In powders, the vehicle is a finely divided solid that is inadmixture with the finely divided active agents according to theinvention. In tablets, the active agent may be mixed with a vehiclehaving the necessary compression properties in suitable proportions andcompacted in the shape and size desired. The powders and tabletspreferably contain up to 99% of the active agents. Suitable solidvehicles include, for example calcium phosphate, magnesium stearate,talc, sugars, lactose, dextrin, starch, gelatin, cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins. Inanother embodiment, the pharmaceutical vehicle may be a gel and thecomposition may be in the form of a cream or the like.

However, the pharmaceutical vehicle may be a liquid, and thepharmaceutical composition is in the form of a solution. Liquid vehiclesare used in preparing solutions, suspensions, emulsions, syrups, elixirsand pressurised compositions. The active agent according to theinvention may be dissolved or suspended in a pharmaceutically acceptableliquid vehicle such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid vehicle can containother suitable pharmaceutical additives such as solubilisers,emulsifiers, buffers, preservatives, sweeteners, flavouring agents,suspending agents, thickening agents, colours, viscosity regulators,stabilisers or osmo-regulators. Suitable examples of liquid vehicles fororal and parenteral administration include water (partially containingadditives as above, for example, cellulose derivatives, preferablysodium carboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, for example, glycols) and theirderivatives, and oils (for example, fractionated coconut oil and arachisoil). For parenteral administration, the vehicle can also be an oilyester such as ethyl oleate and isopropyl myristate. Sterile liquidvehicles are useful in sterile liquid form compositions for parenteraladministration. The liquid vehicle for pressurised compositions can be ahalogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions, which are sterile solutions orsuspensions, can be utilised by, for example, intramuscular,intrathecal, epidural, intraperitoneal, intravenous and particularlysubcutaneous injection. The agent may be prepared as a sterile solidcomposition that may be dissolved or suspended at the time ofadministration using sterile water, saline or other appropriate sterileinjectable medium.

The agents and compositions of the invention may be administered orallyin the form of a sterile solution or suspension containing other solutesor suspending agents (for example, enough saline or glucose to make thesolution isotonic), bile salts, acacia, gelatin, sorbitan monoleate,polysorbate 80 (oleate esters of sorbitol and its anhydridescopolymerized with ethylene oxide) and the like. The agents usedaccording to the invention can also be administered orally either inliquid or solid composition form. Compositions suitable for oraladministration include solid forms, such as pills, capsules, granules,tablets and powders, and liquid forms, such as solutions, syrups,elixirs and suspensions. Forms useful for parenteral administrationinclude sterile solutions, emulsions and suspensions.

It will be appreciated that the invention extends to any nucleic acid orpeptide or variant, derivative or analogue thereof, which comprisessubstantially the amino acid or nucleic acid sequences of any of thesequences referred to herein, including functional variants orfunctional fragments thereof. The terms “substantially the aminoacid/nucleotide/peptide sequence”, “functional variant” and “functionalfragment”, can be a sequence that has at least 40% sequence identitywith the amino acid/nucleotide/peptide sequences of any one of thesequences referred to herein, for example 40% identity with thesequences identified as SEQ ID NOs: 9-69.

Amino acid/nucleotide/peptide sequences with a sequence identity whichis greater than 50%, more preferably greater than 65%, 70%, 75%, andstill more preferably greater than 80% sequence identity to any of thesequences referred to herein are also envisaged. Preferably, the aminoacid/nucleotide/peptide sequence has at least 85% identity with any ofthe sequences referred to, more preferably at least 90%, 92%, 95%, 97%,98%, and most preferably at least 99% identity with any of the sequencesreferred to herein.

The skilled technician will appreciate how to calculate the percentageidentity between two amino acid/nucleotide/peptide sequences. In orderto calculate the percentage identity between two aminoacid/nucleotide/peptide sequences, an alignment of the two sequencesmust first be prepared, followed by calculation of the sequence identityvalue. The percentage identity for two sequences may take differentvalues depending on: (i) the method used to align the sequences, forexample, ClustalW, BLAST, FASTA, Smith-Waterman (implemented indifferent programs), or structural alignment from 3D comparison; and(ii) the parameters used by the alignment method, for example, localversus global alignment, the pair-score matrix used (for example,BLOSUM62, PAM250, Gonnet etc.) and gap-penalty, for example, functionalform and constants.

Having made the alignment, there are many different ways of calculatingpercentage identity between the two sequences. For example, one maydivide the number of identities by: (i) the length of shortest sequence;(ii) the length of alignment; (iii) the mean length of sequence; (iv)the number of non-gap positions; or (iv) the number of equivalencedpositions excluding overhangs. Furthermore, it will be appreciated thatpercentage identity is also strongly length-dependent. Therefore, theshorter a pair of sequences is, the higher the sequence identity one mayexpect to occur by chance.

Hence, it will be appreciated that the accurate alignment of amino acidor nucleic acid sequences is a complex process. The popular multiplealignment program ClustalW [48,49] is a preferred way for generatingmultiple alignments of proteins or DNA in accordance with the invention.Suitable parameters for ClustalW may be as follows: For DNA alignments:Gap Open Penalty=15.0, Gap Extension Penalty=6.66, and Matrix=Identity.For protein alignments: Gap Open Penalty=10.0, Gap ExtensionPenalty=0.2, and Matrix=Gonnet. For DNA and Protein alignments:ENDGAP=−1, and GAPDIST=4. Those skilled in the art will be aware that itmay be necessary to vary these and other parameters for optimal sequencealignment.

Preferably, calculation of percentage identities between two aminoacid/nucleotide/peptide sequences may then be calculated from such analignment as (N/T)*100, where N is the number of positions at which thesequences share an identical residue, and T is the total number ofpositions compared including gaps but excluding overhangs. Hence, a mostpreferred method for calculating percentage identity between twosequences comprises (i) preparing a sequence alignment using theClustalW program using a suitable set of parameters, for example, as setout above; and (ii) inserting the values of N and T into the followingformula: Sequence Identity=(N/T)*100.

Alternative methods for identifying similar sequences will be known tothose skilled in the art. For example, a substantially similarnucleotide sequence will be a sequence which hybridises to a nucleotidesequence encoding a peptide according to the first aspect, or afunctional fragment or functional variant thereof, or their complements,under stringent conditions. By stringent conditions is meant that thenucleotide hybridises to filter-bound DNA or RNA in 3× sodiumchloride/sodium citrate (SSC) at approximately 45° C. followed by atleast one wash in 0.2×SSC/0.1% SDS at approximately 20-65° C.Alternatively, a substantially similar peptide may differ by at least 1,but less than 3, 4, 5, 6, 7, 8, 9 or 10 amino acids from the sequencesshown in SEQ ID NOs: 9-69.

Due to the degeneracy of the genetic code, it is clear that any nucleicacid sequence described herein could be varied or changed withoutsubstantially affecting the sequence of the peptide, polypeptide orprotein encoded thereby, to provide a functional variant thereof.Suitable nucleotide variants are those having a sequence altered by thesubstitution of different codons that encode the same amino acid withinthe sequence, thus producing a silent change. Other suitable variantsare those having homologous nucleotide sequences but comprising all, orportions of, sequence, which are altered by the substitution ofdifferent codons that encode an amino acid with a side chain of similarbiophysical properties to the amino acid it substitutes, to produce aconservative change. For example, small non-polar, hydrophobic aminoacids include glycine, alanine, leucine, isoleucine, valine, proline,and methionine. Large non-polar, hydrophobic amino acids includephenylalanine, tryptophan and tyrosine. The polar neutral amino acidsinclude serine, threonine, cysteine, asparagine and glutamine. Thepositively charged (basic) amino acids include lysine, arginine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. It will therefore be appreciated which aminoacids may be replaced with an amino acid having similar biophysicalproperties, and the skilled technician will know the nucleotidesequences encoding these amino acids.

All of the features described herein (including any accompanying claims,abstract and drawings), and/or all of the steps of any method or processso disclosed, may be combined with any of the above aspects in anycombination, except combinations where at least some of such featuresand/or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodimentsof the same may be carried into effect, reference will now be made, byway of example, to the accompanying diagrammatic drawings, in which:

FIG. 1 shows an amino acid sequence alignment for GAPDH of the mainsepsis-inducing bacteria, GBS, E. coli, S. aureus, S. pneumoniae, K.pneumoniae, P. aeruginosa and N. meningitidis. The multiple alignmentwas obtained from the ClustalW2 server, after submitting the amino acidsequences identified herein as SEQ ID NOs: 1-7 (according to the FASTAformat of the previously indicated UniProt accession numbers). Theresultant % sequence similarities are shown in Table 1.

FIG. 2 provides an example of four surface peptides that can be used ina vaccine of the invention. The table shows the amino acid sequences ofthe four exemplary peptides, and the respective bacteria that possesseach amino acid sequence. Below the table is indicated the surfacelocalisation of the same four peptides in the different bacterialGAPDHs. The peptides are identified herein as Peptides 1-4 (SEQ ID NOs:9-12), and they are used in combination to form the vaccine identifiedherein as Neonatal Vaccine.

FIG. 3 shows an alignment of the amino acid sequence of GAPDH from S.agalactiae, S. pneumoniae, S. aureus and humans. The two boxed areasshow the sequences from which Peptide 1 and Peptide 2 are derived.

FIGS. 4-6 show that neonatal B1 cells are the major producers of IL-10upon stimulus by bacterial GAPDH. Panels A and B of FIG. 4 show IL-10concentration following the stimulation of spleen mononuclear cells(MNC), neutrophils from peripheral blood (PMNC), macrophages (FLM) ordendritic cells (FLDC) derived from the liver and B cells (total), B1cells and B2 cells purified from the spleen of newborn mice withlipopolysaccharide (LPS), rGAPDH or Roswell Park Memorial Institute(RPMI) medium alone.

FIG. 5 shows IL-10 concentration following the stimulation of B1 cellspurified from the spleen of newborn mice with rGAPDH in the presence ofa TLR2 inhibitor (OxPAC) or Toll-like receptor 4 (TLR4) inhibitor(CLI095) as shown.

Panels A and B of FIG. 6 show IL-10 concentration following thestimulation of total cells and B1 cells, respectively, purified from thespleen of newborn mice with rGAPDH, fixed GBS (GBSf) or RPMI mediumalone. Panel C of FIG. 6 shows IL-10 concentration following thestimulation of a co-culture of dendritic cells derived from foetal liverand B1 cells purified from newborn spleen with rGAPDH, GBSf, amonoclonal antibody specific for type I interferon receptor (αIFNAR) orRPMI medium alone. Data depicted in all of the panels for FIGS. 4-6 aremean+SEM of at least two independent experiments.

FIG. 7 shows that TLR2 deficiency improves neonatal survival and confersprotection to bacterial sepsis. The survival of newborn mice challengedwith S. aureus strain NEWMAN (panel A) or E. coli strain IHE3034 (panelB) is shown. Both wild-type and TLR2^(−/−) mice were included in thestudy. Results represent data pooled from at least two independentexperiments. The numbers between parentheses represent the number ofanimals that survived the different infectious challenges versus thetotal number of infected animals. Statistical differences (P values)between TLR2-deficient pups versus controls are indicated.

FIG. 8 shows that blocking IL-10 signaling protects newborns frombacterial sepsis. The survival of newborn mice challenged with E. colistrain IHE3034 (panel A) or S. aureus strain NEWMAN (panel B) followingan injection of monoclonal antibodies specific for the mouse IL-10receptor (anti-11,10R) or isotype-matched control antibodies (isotypeIgG/control) is shown. Results represent data pooled from threeindependent experiments. The numbers between parentheses represent thenumber of animals that survived the different infectious challengesversus the total number of infected animals. Statistical differences (Pvalues) between anti-IL-10R-treated pups versus controls are indicated.

FIG. 9 shows that GAPDH secretion is a shared virulence mechanism.rGAPDH, which was used as a positive control, is shown in lane 1. Lanes2-6 show an equivalent band for the indicated pathogens (NEM316 being astrain of GBS). The data are representative of five independentexperiments.

FIG. 10 shows that antibodies elicited with rGAPDH protect newborn micefrom infection by sepsis-inducing bacteria other than GBS. The survivalof mice pups challenged with S. pneumonia strain Tigr4 (Panel A), E.coli strain IHE3034 (Panel B) and S. aureus strain NEWMAN (Panel C)following an injection of rGAPDH-induced antibodies (anti-rGAPDH IgG) orcontrol antibodies (Control IgG) is shown. Results represent data pooledfrom two independent experiments. The numbers between parenthesesrepresent the number of animals that survived the various infectiouschallenges versus the total number of infected animals. Statisticaldifferences (P values) between immunised versus control groups areindicated.

FIG. 11 shows that bacterial GAPDH induces IL-10 production in humanmononuclear cells. Panels A and B show IL-10 concentration following thestimulation of human mononuclear cells separated from cord-blood (panelA) or peripheral blood (panel B) with rGAPDH, a TLR2 inhibitor (TLR2 in)or RPMI medium alone. Data depicted in the figure are mean+SEM of atleast two independent experiments.

FIG. 12 shows an alignment of the amino acid sequence of GAPDH from E.coli and humans. The boxed area shows Peptide 3 from FIG. 2.

FIG. 13 shows an alignment of the amino acid sequence of GAPDH from P.aeroginosa and humans. The boxed area at amino acid 23 shows a peptidefor use in the invention (SEQ ID NO: 62). The boxed area at amino acid59 shows Peptide 4 from FIG. 2.

FIGS. 14, 15 and 16 show that antibodies elicited with a vaccine of theinvention react with bacterial GAPDH. rGAPDH, which was used as apositive control, is shown in lane 1 of each gel. Lanes 2-6 in FIG. 14and lane 2 in FIGS. 15 and 16 show an equivalent band for the indicatedpathogens. The data are representative of two independent experiments.

FIG. 17 shows that antibodies elicited with Neonatal Vaccine protectnewborn mice from GBS infection. The survival of mice pups challengedwith GBS NEM316 following an injection of Neonatal Vaccine-inducedantibodies (IgG) or control IgG is shown. Results represent data pooledfrom two independent experiments. The numbers between parenthesesrepresent the number of animals that survived the infectious challengeversus the total number of infected animals. Statistical differences (Pvalues) between immunised versus control groups are indicated.

FIG. 18 shows that antibodies elicited with Neonatal Vaccine protectnewborn mice from bacterial sepsis. The survival of newborn micechallenged with S. pneumoniae strain Tigr4 (panel A), E. coli strainIHE3034 (panel B) or S. aureus strain NEWMAN (panel C) following aninjection of Neonatal Vaccine-induced antibodies (IgG) or control IgG isshown. Results represent data pooled from at least two independentexperiments. The numbers between parentheses represent the number ofanimals that survived the different infectious challenges versus thetotal number of infected animals. Statistical differences (P values)between immunised versus controls are indicated.

FIG. 19 shows that the therapeutic use of anti-GAPDH antibodies canefficiently treat GBS-induced sepsis. The survival of newborn micechallenged with GBS NEM316 and subsequently receiving NeonatalVaccine-induced antibodies (IgG), control IgG or saline is shown.Results represent data pooled from two independent experiments.

The numbers between parentheses represent the number of animals thatsurvived the infectious challenge versus the total number of infectedanimals. Statistical differences (P values) between immunised versuscontrols are indicated.

FIG. 20 shows that antibodies elicited with Neonatal Vaccine protect oldmice against lethal GBS infection. The survival of old mice challengedwith GBS following an injection of Neonatal Vaccine-induced antibodies(Neonatal Vaccine-IgG) or control IgG (‘sham-immunized’) is shown.Results represent data pooled from two independent experiments. Thenumbers between parentheses represent the number of animals thatsurvived the infectious challenge versus the total number of infectedanimals. The statistical difference (P value) between immunised versuscontrol groups is indicated.

FIG. 21 shows that antibodies elicited with Neonatal Vaccine protectnon-obese diabetic (NOD) mice against lethal GBS infection. The survivalof NOD mice challenged with GBS following injections of NeonatalVaccine-induced antibodies (Neonatal Vaccine-IgG) or control IgG(‘sham-immunized’) is shown. Results represent data pooled from twoindependent experiments. The numbers between parentheses represent thenumber of animals that survived the infectious challenge versus thetotal number of infected animals. The statistical difference (P value)between immunised versus control groups is indicated.

EXAMPLES

The materials and methods employed in the studies described in theExamples were as follows, unless where otherwise indicated:

Mice

Six- to eight-week-old male and female BALB/c, C57BL/6, andTLR2-deficient C57BL/B6.129-Tlr2^(tm1Kir/J) (TLR2^(−/−)) mice, and oldC57Bl/6 mice (over 16 months), were purchased from The JacksonLaboratory. New Zealand White rabbits and eight-week-old non-obesediabetic (NOD) mice were purchased from Charles River Laboratories.Animals were kept at the animal facilities of the Institute Abel Salazarduring the time of the experiments. All procedures were performedaccording to the European Convention for the Protection of VertebrateAnimals used for Experimental and Other Scientific Purposes (ETS 123)and 86/609/EEC Directive and Portuguese rules (DL 129/92). All animalexperiments were planned to minimise animal suffering.

Bacteria

The bacteria used in the studies are listed in Table 4 below. Allstrains were clinical isolates obtained from infected newborns. E. coli,S. aureus, P. aeruginosa, GBS and S. pneumoniae were kindly provided byProfessor Patrick Trieu Cuot from Pasteur Institute, Paris, France; K.pneumoniae and N. meningitidis were provided by the MicrobiologyDepartment of Hospital Geral de Santo Antonio, Porto, Portugal. GBS andS. pneumoniae were grown in Todd-Hewitt broth or agar (DifcoLaboratories) containing 0.001 mg/mL of colistin sulphate and 0.5 μg/mLof oxalinic acid (Streptococcus Selective Supplement, Oxoid). E. coli,P. aeruginosa, MenB and S. aureus were cultured on Todd-Hewitt broth oragar medium. Bacteria were grown at 37° C.

TABLE 4 Bacteria used in the studies described in the Examples BacteriaStrain Escherichia coli IHE 3034 Staphylococcus aureus NEWMANPseudomonas aeroginosa PAO4 Streptococcus agalactiae, GBS NEM316Streptococcus pneumoniae Tigr4 Neisseria meningitidis Serogroup B (MenB)

Antibody Treatments

Antibody treatments were performed in newborn BALB/c mice (up to 48 hold) 12 h prior to GBS infection, and in old C57Bl/6 mice (over 16months) and NOD mice 24 h prior to GBS infection. For passiveimmunisations, pups were intraperitoneally injected with 100 μg ofanti-rGAPDH IgG antibodies. Control animals received the same amount ofcontrol IgG antibodies. For IL-10 signaling blocking, 100 μg ofanti-IL10R antibodies (1B1.3a, Schering-Plough Corporation) wereadministered intraperitoneally and control animals received the sameamount of matched isotype control antibody. Regarding the therapeuticuse of anti-GAPDH antibodies, mice pups were treated with 100 μg ofanti-GAPDH IgG (or the respective control IgG) 6 h after infection.

Neonatal Mouse Model of Bacterial Infection

Neonatal (48 h old), BALB/c, C57BL/6 wild-type or TLR2^(−/−) mice wereinfected subcutaneously with the indicated inoculum of the bacteria in amaximum volume of 40 μl. Newborns were kept with their mothers duringthe entire time of the experiment. Survival curves were determined overa 12-day experimental period.

rGAPDH

rGAPDH was produced and purified as previously described [41].

Purification of Anti-GAPDH IgG

Adult mice or rabbits were immunised twice with 25 μg of rGAPDH in aPBS/alum suspension with a three-week interval between doses. Sera werecollected 10 days after the second immunisation. Pooled serum sampleswere applied to a Protein G HP affinity column (HiTrap, GE HealthcareBio-Sciences AB) and purified IgG antibodies were then passed through anaffinity column with immobilised rGAPDH (Hi-trap NHS-activated HP, GEHealth-care Bio-Sciences AB). Control IgGs were obtained from sera ofmice or rabbits sham-immunised with a PBS/alum suspension and purifiedon a Protein G HP affinity column. Purified IgG antibody fractions werefurther equilibrated in PBS and stored at −80° C. in frozen aliquots.

Spleen Total Cell Cultures

Cells from the spleen of newborn mice (up to 48 h old) were obtained bygently teasing the organ in RPMI 1640 supplemented with penicillin (100IU/ml), streptomycin (50 μg/ml), 2-ME (0.05 M), and 10% foetal bovineserum (FBS) (Sigma-Aldrich)—complete RPMI (cRPMI). Cells were thendistributed in 96-well plates (1×10⁶ cells/well) and cultured for 12 hat 37° C. in a humidified atmosphere containing 5% carbon dioxide, withthe medium alone, medium containing 2.5 μg/ml LPS, medium containing 25μg/ml of rGAPDH, medium containing 1 μg/mL of the TLR2 agonist, PAM3CSK4(Invivogen). For the experiments with the TLR inhibitors, OxPAC (TLR2inhibitor) and CLI095 (TLR4 inhibitor) (both from Invivogen) were usedat a concentration of 10 μg/mL.

B Cell Purification

B cells were purified from the spleen of neonatal mice (prepared asmentioned above) by magnetic cell sorting using a Mouse B cellPurification Kit (Miltenyi Biotech) according to manufacturer'sinstructions.

Cd5⁺ B Cell Purification

B1 cells were purified from the spleen of neonatal mice (prepared asmentioned above) by magnetic cell sorting, using a Mouse B1 cellPurification Kit (Miltenyi Biotech) according to manufacturer'sinstructions.

Neonatal Liver-Derived Macrophages

Macrophages were obtained from the liver of one-day old mice. Liverswere removed under aseptic conditions and homogenised in Hanks' balancedsalt solution (HBSS). The resulting cell suspension was centrifuged at500×g and resuspended in cRPMI supplemented with 10% L929 cellconditioned medium. To remove fibroblasts or differentiated macrophages,cells were cultured, on cell culture dishes, overnight at 37° C. in a 5%carbon dioxide atmosphere. Then, non-adherent cells were collected withwarm cRPMI, centrifuged at 500×g, distributed in 96-well plates at adensity of 1×10⁵ cells/well, and incubated at 37° C. in a 5% carbondioxide atmosphere. Four days after seeding, 10% of L929 cellconditioned medium was added, and the medium was renewed on the seventhday. After 10 days in culture, cells were completely differentiated intomacrophages. This method allows for the differentiation of a homogenousprimary culture of macrophages that retain the morphological,physiological and surface markers characteristics of these phagocyticcells [50].

Neonatal Liver-Derived Dendritic Cells

Dendritic cells were obtained from the liver of one-day old mice. Liverswere removed under aseptic conditions and homogenised in HBSS. Theresulting cell suspension was centrifuged at 500×g and resuspended incRPMI supplemented with 30 ng/ml of granulocyte macrophagecolony-stimulating factor (GM-CSF) (Immunotools) (Primary DC media). Toremove fibroblasts or differentiated macrophages, cells were cultured,on cell culture dishes, overnight at 37° C. in a 5% carbon dioxideatmosphere. At day 3, 75% of the medium (along with non-adherent cells)was removed, and Primary DC media was added. At day 6, cells wereremoved from the plate by gently pipetting media up and down against thebottom of the plate to gently dislodge non-adherent cells. After severalminutes of this, the cell mixture was transferred to 50 mL polystyrenetubes. Cells were then centrifuged at 500×g for 5-7 min and re-suspendedin Primary DC media. The cells were counted and plated at aconcentration of 5×10⁵ cells/well. For the co-culture experiments, 5×10⁴dendritic cells were plated per well. In the co-culture experiments, andwhere indicated, 20 μg/mL of a monoclonal antibody specific for Type Iinterferon receptor (anti-IFNAR) (Biolegend) was used.

Purification of Blood Neutrophils

For neutrophil isolation, blood was collected from retro-orbitalbleeding of neonatal mice (up to 48 h old) and diluted 1:2 in HBSScontaining BSA (0.1% w/v) and glucose (1% w/v). Cells were pelleted, anderythrocytes were removed by hypotonic lysis. The blood preparation wassuspended in Dulbecco's PBS (GIBCO), layered on a three-layer Percoll(GE-Healthcare) gradient (80, 65, and 55% in Dulbecco's PBS), andcentrifuged at 1200×g for 30 min at 10° C. Mature neutrophils wererecovered at the interface of the 65 and 80% fractions, and purity was85%, as determined by FACS analysis, using anti-Ly6G antibodies(Biolegend). Isolated neutrophils were plated on 96-well plates andstimulated for 12 h as indicated.

IL-10 Quantification

IL-10 from newborn or adult cell cultures was quantified by ELISA (R&DSystems), according to the manufacturer's instructions.

Human Blood Samples

Human blood samples were obtained at Hospital Geral de Santo Antónioafter informed approval. For the isolation of mononuclear cells, 5 mlaliquots of total blood diluted 1:2 in RPMI 1640 were layered on 2.5 mlof Histopaque (Sigma-Aldrich) and centrifuged at 1000 g for 20 min atroom temperature. The cells were then gently removed from themedium-Histopaque interface, transferred to a sterile container, andwashed in 10 ml of cRPMI. The isolated mononuclear cells werere-suspended in cRPMI, plated at a concentration of 5×10⁵ cells/well andstimulated with 25 μg/mL of rGAPDH, with 10 μg/mL of OxPAC or withmedium alone (RPMI) for 12 h at 37° C. with 5% carbon dioxide.

Neonatal Vaccine

Peptides 1-4 (SEQ ID NOs: 9-12) were conjugated with KLH or OVA ascarrier proteins. For the immunisation protocols, 20 μg of each peptideconjugated with the carrier protein was injected intraperitoneally infemale BALB/c mice. Alum was used as adjuvant in a 1:20 PBS suspension.Adult female BALB/c mice were immunised three times with a three-weekinterval between doses. 10 days after the last immunisation, blood wascollected and the “Neonatal Vaccine” anti-serum was obtained after bloodclotting at 4° C. for 24 hours.

The same immunisation protocol was used in rats for the N. meningitidiswork (Example 8).

Example 1—A Sub-Population of Neonatal B Cells is Responsible for IL-10Production Upon Bacterial GAPDH Stimulus

Previous published information revealed the role of GAPDH in disablingthe neonatal immune system to combat GBS infections [24]. Although arole for IL-10 was already unveiled in the mechanism of GAPDH-inducedimmunosuppression, the cellular mechanism remained unknown. In order touncover which cellular population(s) was contributing to early IL-10production observed in neonatal GBS infections, different leukocytepopulations were purified from neonatal mice and treated in vitro withrGAPDH from GBS.

Materials and Methods

Specifically, and as described above, dendritic cells and macrophageswere obtained from neonatal liver precursors, B cells and mononuclearcells were obtained from neonatal spleens and neutrophils were purifiedfrom neonatal peripheral blood. A more refined separation of B cellsobtained from neonatal spleen, based on surface expression of CD5,allowed the separation of B1 (CD5+) cells.

The different leukocyte populations were stimulated in vitro with 0.5μg/mL of LPS (as a positive control; LPS is a structural microbialantigen known to induce polyclonal B cell activation), 25 μg/mL ofrGAPDH or RPMI medium alone (as a negative control) for 12 h at 37° C.with 5% carbon dioxide. In all conditions 5×10⁵ cells/well were used,except for the separated B cell study, where 2.5×10⁵ cells/well wereused.

After incubation of the cells, IL-10 concentration was measured in thesupernatants as described above.

At least two independent experiments were performed in each case.

Results

As observed in FIG. 4A, when comparing the ability of mononuclear cells,neutrophils, macrophages, dendritic cells and total B cells to produceIL-10 upon GAPDH stimulus, only B cells retained the ability to producesignificant amounts of IL-10.

Following the separation of neonatal B cells, the inventors observedthat B1 cells retained the ability to produce IL-10 while B2 cellsproduced only traceable amounts of this cytokine (FIG. 4B).

Discussion

This study indicates that neonatal B1 cells are the main source of IL-10upon bacterial GAPDH stimulus.

Example 2—TLR2 is the Surface Receptor for Bacterial GAPDH

In order to establish the cellular receptor responsible for bacterialGAPDH recognition and induction of IL-10 expression, the inventorscompared the ability of GAPDH to induce IL-10 production in cultures ofpurified B1 cells in the presence of specific inhibitors of differentpattern recognition receptors.

Materials and Methods

B1 cells were purified from the spleen of newborn mice as describedabove.

2.5×10⁵ B1 cells/well were stimulated in vitro with 25 μg/mL of rGAPDHin the presence of 10 μg/ml of TLR2 or TLR4 inhibitors for 12 h at 37°C. with 5% carbon dioxide. The TLR2 and TLR4 inhibitors used were OxPACand CLI095, respectively.

After incubation of the cells, IL-10 was quantified in the supernatantsas described above.

At least two independent experiments were performed.

Results

The inventors found that GAPDH-induced IL-10 production was completelyabrogated in the presence of a TLR2 inhibitor (FIG. 5).

Discussion

This result indicates that bacterial GAPDH acts on B1 cells through TLR2in order to induce IL-10 production.

Example 3—TLR2 Deficiency Improves Neonatal Survival and ConfersProtection to Bacterial Sepsis

This study aimed to confirm the importance of TLR2 as a receptor forGAPDH, and a cause for neonatal susceptibility to sepsis.

Materials and Methods

48 hours after birth, newborn wild-type and TLR2^(−/−) mice wereinfected subcutaneously with 5×10⁵ CFU of S. aureus strain NEWMAN orwith 500 CFU of E. coli strain IHE3034. Survival of the mice followinginfection was monitored on a daily basis.

At least two independent experiments were performed.

Results

Wild-type mice were unable to survive infection with the indicatedbacteria beyond 48 hours post-infection (FIG. 7). In contrast, themajority of TLR2^(−/−) mice were still alive at 12 days after infection.

Discussion

The results shows that TLR2-deficient neonatal mice have increasedsurvival against challenging infections with E. coli and S. aureuscompared to wild-type mice. TLR2 thus plays an important role inneonatal susceptibility to sepsis; that is to say, TLR2 deficiencyimproves neonatal survival and confers protection to bacterial sepsis.In addition to the results obtained in Example 2, these data thusconfirm the importance of TLR2 as a receptor for GAPDH, across thedifferent species of sepsis-inducing bacteria.

Example 4—Type I Interferon Production by Dendritic Cells Induced byBacteria Synergises with GAPDH to Increase IL-10 Production on B1 Cells

This study aimed to identify whether B1 cells are assisted in theproduction of IL-10 upon GAPDH recognition by other leukocytepopulations, and in what capacity.

Materials and Methods

Total spleen cells were obtained from newborn mice, and B1 cells werepurified from the total spleen cell population, as described above.

The different spleen cell populations were stimulated in vitro with 25μg/mL of rGAPDH, 10⁷ cells of GBS fixed in isopropanol (GBSf) or withRPMI medium alone for 12 h at 37° C. with 5% carbon dioxide. In allconditions 5×10⁵ cells/well were used, except for the purified B1 cellstudy where 2.5×10⁵ cells/well were used.

Dendritic cells were derived from foetal liver as described above. Thedendritic cells were co-cultured with 2.5×10⁵ of the B1 cells purifiedfrom newborn spleen in a 1:10 ratio and stimulated with 25 μg/mL ofrGAPDH, 10⁷ cells of GBSf, 20 μg/mL of anti-IFNAR or with RPMI mediumalone for 12 h at 37° C. with 5% carbon dioxide.

After incubation of the different cell types, IL-10 was quantified inthe supernatants as described above.

At least two independent experiments were performed in each case.

Results & Discussion

The ability of GAPDH to induce IL-10 production in total spleen cellswas strongly increased in the presence of fixed bacteria (FIG. 6A).Nevertheless, this effect was lost in purified B1 cells, where addingfixed bacteria did not increase the IL-10 production induced by GAPDH(FIG. 6B).

This result indicates that different leukocyte population(s) other thanB1 cells are stimulated by bacterial antigens and help B1 cells toproduce IL-10 upon GAPDH recognition.

The co-culture study enabled understanding of the role of othersub-populations of leukocytes in the influence of B1 cells to produceIL-10. The inventors observed that in the presence of dendritic cells,B1 cells produced elevated amounts of IL-10 when stimulatedsimultaneously with GAPDH plus GBSf (FIG. 6C). Interestingly, thiseffect was abrogated when type I interferon signalling was blocked (FIG.6C).

This result indicates that upon bacterial recognition, dendritic cellsproduce type I interferon that increase IL-10 production in B1 cellsstimulated with GAPDH.

Example 5—Early IL-10 Production is a Generalised Mechanism Used bySepsis-Inducing Bacteria to Colonise the Neonatal Host

The ability to produce high amounts of IL-10 was demonstrated to be themain reason for the susceptibility of neonates against GBS infections[24]. The present study aimed to investigate whether the same happens inneonatal infections caused by bacteria other than GBS, specifically E.coli or S. aureus. Together with GBS, E. coli and Staphylococcal spp.are responsible for up to 87% of the cases of sepsis in human neonates.

The study also aimed to investigate whether other bacteria also possessextracellular GAPDH, as an indication of a generalised IL-10-dependentmechanism used by sepsis-inducing bacteria to colonise the neonatalhost.

Materials and Methods

Neonatal mice were treated with blocking antibodies specific for themouse IL-10 receptor (anti-11,10R) before challenge with E. coli or S.aureus, as follows.

Newborn mice were intraperitoneally injected with 100 μg of anti-11,10Rmonoclonal antibodies or 100 μg of isotype control IgG as describedabove. 12 h later the mice were challenged subcutaneously with 500 CFUof E. coli strain IHE3034 or with 5×10⁵ CFU of S. aureus strain.Survival of the mice following infection was monitored on a daily basis.

Three independent experiments were performed.

To investigate whether other sepsis-inducing pathogens also possessextracellular GAPDH, extracellular proteins from culture supernatants ofpathogens of interest were obtained, separated by SDS-PAGE and analysedby western-blot using anti-rGAPDH antibodies, as follows.

Cultures of GBS strain NEM316, P. aeruginosa, S. pneumoniae, S. aureusand E. coli were prepared as described above, and extracellular proteinspurified from the culture supernatants in accordance with standardprocedures. SDS-PAGE and Western-blot analysis were performed accordingto standard procedures using anti-rGAPDH antibodies (IgG) obtained fromrGAPDH-immunised rabbits as described above. rGAPDH was used as apositive control.

Five independent experiments were performed.

The effect of neutralising GAPDH secreted by these bacteria, to assesswhether these pathogens also use GAPDH secretion as a virulencemechanism, was investigated as follows.

Mice pups were intraperitoneally injected with 80 μg of anti-rGAPDHantibodies (IgG) or 80 μg of control IgG as described above. 12 h laterthe mice were challenged subcutaneously with 5×10⁶ CFU of S. pneumoniaestrain Tigr4, 500 CFU of E. coli strain IHE3034 or 5×10⁵ CFU of S.aureus strain NEWMAN. Survival of the mice following infection wasmonitored on a daily basis.

Two independent experiments were performed.

Results

Interestingly, blocking IL-10 signalling significantly improved survivalof neonates to infections caused by E. coli and S. aureus, when comparedwith pups that received isotype-matched control antibodies (FIG. 8).

Other sepsis-inducing bacteria were also shown to possess extracellularGAPDH (FIG. 9). Neutralisation of this secreted GAPDH using anti-rGAPDHantibodies was shown to protect newborn mice from infection by S.pneumoniae, E. coli and S. aureus (FIG. 10A-C, respectively).

Discussion

The results indicate that the mechanism observed for the susceptibilityof neonates against GBS-induced sepsis is transversal to othersepsis-inducing bacteria. Although IL-10 data for neonatal infectionscaused by E. coli and S. aureus are provided here, the fact that otherbacteria also secrete GAPDH is a strong indicator that the propensity ofneonates to produce high amounts of IL-10 in response to bacterial GAPDHis a global mechanism used by different bacterial pathogens, which leadsto the development of sepsis. This result validates the fact that aGAPDH-based vaccine against GBS will also be viable against othersepsis-inducing bacteria too.

Example 6—GAPDH-Induced IL-10 Production is a Mechanism Conserved inHuman Cells

This study aimed to investigate whether human cells also produce IL-10in response to GAPDH.

Materials and Methods

Mononuclear cells were separated from human cord-blood or adultperipheral blood as described above.

The cells were stimulated in vitro with 25 μg/mL of rGAPDH, 10 μg/mL ofOxPAC (a TLR2 inhibitor) or RPMI medium alone for 12 h at 37° C. with 5%carbon dioxide.

After incubation of the cells, IL-10 was quantified in the supernatantsas described above.

At least two independent experiments were performed.

Results

In agreement with what was observed in neonatal mice, the stimulation ofmononuclear cells purified from human cord-blood or adult peripheralblood with rGAPDH induced the production of high amounts of IL-10 (FIG.11A and FIG. 11B, respectively).

Interestingly, GAPDH-induced IL-10 production in human leukocytes wascompletely abrogated in the presence of a TLR2 inhibitor.

Discussion

This result shows that the mechanism for IL-10 production induced byGAPDH in mouse cells is also true for humans.

Moreover, the fact that the same virulence mechanism studied in mice canbe readily translated to humans strongly supports the use of mice as anexcellent model to study bacterial sepsis in man.

Example 7—Production of Neonatal Vaccine

Based on their discovery that the propensity of neonates to produce highamounts of IL-10 in response to bacterial GAPDH is a global mechanismused by different bacterial pathogens, the inventors set out to producea vaccine against such pathogens using GAPDH-derived peptides as theantigen.

Materials and Methods

Neonatal Vaccine was prepared as described above.

Results

Vaccines of the invention are composed from surface peptides of GAPDHfrom the different sepsis-inducing bacteria, which have amino acidsequences that are absent from human GAPDH. As such, the inventors havedeveloped vaccines composed of peptides belonging to conserved sequencesof microbial GAPDH that are not shared by human GAPDH.

FIG. 2 includes a table identifying the amino acid sequences of fourexemplary peptides that were found in the present study, using the abovemethod, and the respective bacteria that possess each amino acidsequence. Below the table are images showing the surface localisation ofthe same four peptides in the different bacterial GAPDHs.

FIG. 3 illustrates how two of the peptides (identified as Peptides 1 and2 in FIGS. 2 and 3) have amino acid sequences which are conservedamongst certain bacterial species, but not in humans.

FIGS. 12 and 13 illustrate how two further peptides from E. coli and P.aeruginosa (identified as Peptides 3 and 4, respectively, in FIG. 2)have (bacterially conserved) amino acid sequences which are not found inman.

Peptides 1-4 were used in combination in the preparation of a preferredvaccine of the invention, referred to herein as Neonatal Vaccine. Thus,Neonatal Vaccine is suitable for use against all of the bacteria listedin the table in FIG. 2.

Peptides 1-4, however, are mere examples; that is to say, other peptidesthat are suitable for use in a vaccine of the invention can beidentified by sequence alignment in the same way as set out above. Anyother amino acid sequence can be used from GAPDHs of the referredpathogens. As explained herein, however, it is preferable to avoid anysequences that are also found in man, so as to avoid any autoimmunepathologies.

As described herein, any number of peptides, in any combination, can beused instead of Peptides 1-4 of Neonatal Vaccine. That is to say, thenumber and identity of peptides that constitute a vaccine of theinvention can vary.

Discussion

As explained herein, bacterial GAPDH plays a role in causingimmunosuppression in neonates and immunocompromised hosts and promotingbacterial sepsis. The vaccines described herein, including the specificNeonatal Vaccine described in this example, are thus directed to protectsusceptible hosts (including neonates, the elderly and other suchimmunocompromised individuals) from infections caused by GBS, E. coli,Staphylococcus spp., S. pneumoniae, K. pneumoniae and Pseudomonas spp.The approach taken by the inventors allows the possibility to “tailor” avaccine of the invention for any sepsis-inducing bacteria, simply byselecting the surface-exposed peptides of its GAPDH that are absent fromhuman GAPDH.

Example 8—Antibodies Elicited with Neonatal Vaccine React with BacterialGAPDH

This study aimed to show that the vaccine produced in Example 7 could beused to produce antibodies that recognise bacterial GAPDH.

Materials and Methods

Mice and rats were immunised with Neonatal Vaccine as described above.

Cultures of GBS strain NEM316, P. aeruginosa, S. pneumoniae, S. aureus,E. coli, K. pneumoniae and MenB were prepared as described above, andextracellular proteins purified from the culture supernatants inaccordance with standard procedures. SDS-PAGE and Western-blot analysiswere performed according to standard procedures using anti-GAPDHantibodies (IgG) obtained from the rGAPDH-immunised mice and rats asdescribed above. rGAPDH was used as a positive control.

Two independent experiments were performed.

Results

As shown in FIGS. 14-16, antibodies purified from mice and ratsimmunised with Neonatal Vaccine react with extracellular GAPDH from thedifferent bacteria.

FIG. 15 (showing results from K. pneumoniae) reveals two bands that arerecognised by anti-GAPDH antibodies elicited with Neonatal Vaccine.Interestingly, the band of ˜45 KDa corresponds to exactly the samemolecular weight of GBS GAPDH. The other band (˜35 KDa) corresponds tothe predicted molecular weight of K. pneumoniae GAPDH(http://www.uniprot.org/uniprot/C4X7S6).

FIG. 16 (showing results from MenB) reveals a band of ˜37 kDa, whichcorresponds to the predicted molecular weight of MenB GAPDH [51].

Discussion

This study shows that antibodies elicited with Neonatal Vaccinerecognise GAPDH from GBS strain NEM316, P. aeruginosa, S. pneumoniae, S.aureus, E. coli, K. pneumoniae and MenB. These data therefore provideproof-of-concept that bacterial peptide sequences in common can be usedin a vaccine to recognise bacterial GAPDH.

Although only serotype B of N. meningitidis has been tested here,similar results would be expected for all other serotypes of thisbacterium. In this regard, GAPDHs from the different serotypes of N.meningitidis share high (97.668%) homology(http://www.uniprotorg/align/A20150610146R80D4XR) and antibodieselicited with Neonatal Vaccine would therefore be expected to recogniseGAPDH from them all. It is consequently believed that the vaccinesdescribed herein are advantageous for all the serotypes of N.meningitidis.

The result illustrated in FIG. 15 also shows that, interestingly, K.pneumoniae may possess two isoforms of GAPDH.

Example 9—Neonatal Vaccine Protects Neonates from GBS Infection

This study aimed to show that the antibodies produced in Example 8 couldbe used to protect newborn mice from GBS infection.

Materials and Methods

Mice pups were intraperitoneally injected with 80 μg of NeonatalVaccine-induced IgG or 80 μg of control IgG as described above. 12 hlater the mice were challenged subcutaneously with 5×10⁶ CFU of GBSNEM316. Survival of the mice following infection was monitored on adaily basis.

Two independent experiments were performed.

Results

Maternal vaccination with rGAPDH (whole protein) has previously provento be an efficient strategy to prevent neonatal infections caused by GBS[24]. However, when antibodies elicited with Neonatal Vaccine were usedfor passive immunisations of pups before GBS infection, the protectionwas even more effective. Indeed, the protection conferred with NeonatalVaccine was 100% (FIG. 17).

Discussion

This result shows that the new approach used to develop the vaccines ofthe invention, including Neonatal Vaccine, (i.e. using select peptidesequences, as described herein, instead of the whole protein) directsthe immune system of neonates to a more robust and specific responsetowards sepsis-inducing agents, exemplified by GBS, compared to thatpreviously described.

This result also shows that the immunity provided by Neonatal Vaccine isreproducible (100% effective), which is clearly advantageous.

Although this study only looked at GBS infection, it is understood thatthe same result would be observed upon infection by othersepsis-inducing bacteria (not least because Example 8 shows thatantibodies elicited with Neonatal Vaccine recognise GAPDH from GBSstrain NEM316, P. aeruginosa, S. pneumoniae, S. aureus, E. coli, K.pneumoniae and N. meningitidis (as exemplified by MenB)).

Moreover, the results presented in FIG. 17 provide evidence thatmaternal vaccination with Neonatal Vaccine (or, therefore, maternaltreatment with the anti-GAPDH antibodies of the invention), willsignificantly reduce stillbirths and premature births caused byintra-vaginal GBS infection. Indeed, as discussed above in connectionwith the twelfth aspect of the invention, antibodies raised against apeptide, fragment, variant or vaccine of the invention can pass to anunborn baby across the mother's placenta. In addition, and as shown inFIG. 17, the protection against GBS conferred on pups followingimmunisation with Neonatal Vaccine-induced antibodies was 100%.

Owing to the high sequence similarity and functional homology betweenGAPDH of GBS and the other sepsis-inducing bacteria as described herein,the presented data also indicate that Neonatal Vaccine will effectivelyprevent stillbirths and pre-term births caused by other sepsis-inducingbacteria too. This is an important finding, as bacterial infections areresponsible for approximately 650,000 stillbirths per year worldwide[52,53]. In addition, about 50% of preterm births at less than 32 weeksof gestation are also caused by bacterial infections [9,14,53-56]. Thevast majority are caused by maternal commensal bacteria that ascend fromthe vaginal tract into the amniotic fluid. GBS, E. coli and K.pneumoniae are the most common pathogens found in autopsies ofstillbirth babies caused by ascending bacterial infections.

Example 10—Neutralisation of Bacterial GAPDH is a Global Approach toProtect Neonates from Bacterial Sepsis

This study aimed to extend the work described in Example 9, byinvestigating whether antibody-mediated neutralisation of bacterialGAPDH could prevent neonatal infections caused by the other relevantsepsis-inducing bacteria.

Materials and Methods

Mice pups were intraperitoneally injected with 80 μg of NeonatalVaccine-induced IgG or 80 μg of control IgG as described above. 12 hlater the mice were challenged subcutaneously with 10⁷ CFU of S.pneumoniae strain Tigr4, 500 CFU of E. coli strain IHE3034 or 5×10⁵ CFUof S. aureus strain NEWMAN. Survival of the mice following infection wasmonitored on a daily basis.

Results

As shown in FIG. 18, the use of antibodies elicited with NeonatalVaccine in passive immunisations of neonates significantly improvesurvival upon bacterial challenge. Here are shown results for S.pneumoniae, E. coli and S. aureus (see FIG. 18A-C, respectively).

Discussion

As discussed herein, currently there is no available vaccine directed toany of the most relevant sepsis-inducing bacteria. Presented here aredata demonstrating that antibody-mediated neutralisation of bacterialGAPDH prevents neonatal infections caused by the most relevantsepsis-inducing bacteria.

Example 11—Therapeutic Administration of Neonatal Vaccine IgG AntibodiesProtects Newborn Mice from GBS Infection

This study aimed to investigate whether antibodies elicited withNeonatal Vaccine could treat an existing neonatal infection caused bysepsis-inducing bacteria.

Materials and Methods

Neonatal Vaccine IgG, control IgG (80 μg) or saline solution (0.9% NaCl)were intraperitoneally injected into mice pups (up to 48 h old) 6 hafter subcutaneous infection with 5×10⁶ GBS NEM316 CFU. At the time oftreatment all mice presented clear signs of infection, assessed byintense rash at the site of infection. Survival of the mice followinginfection was monitored on a 12-hourly basis.

Results

As shown in FIG. 19, only the mice that received NeonatalVaccine-induced IgG antibodies were able to survive GBS infection. Infact, treatment of mice pups with anti-GAPDH IgG antibodies after GBSinfection resulted in complete survival of the animals. In contrast,none of the controls survived the infection.

Discussion

As discussed herein, the current treatment available for neonatal sepsisis based only on antibiotic administration. Presented here are datademonstrating that antibodies induced by Neonatal Vaccine can be used totreat existing neonatal infections caused by GBS, one of the mostrelevant sepsis-inducing bacteria.

Although this study only looked at GBS infection, it is understood thatthe same result would be observed upon infection by othersepsis-inducing bacteria (not least because Example 8 shows thatantibodies elicited with Neonatal Vaccine recognise GAPDH from GBSstrain NEM316, P. aeruginosa, S. pneumoniae, S. aureus, E. coli, K.pneumoniae and N. meningitidis (as exemplified by MenB)).

The peptides, fragments and variants of the first aspect of theinvention thus have significant utility in creating a variety of usefuland much-needed antibody-based therapeutics for the indicated patientpopulations.

Example 12—Neonatal Vaccine Protects Old Mice from GBS Infection

This study aimed to show that the antibodies produced in Example 8 couldbe used to protect old mice from GBS infection.

Materials and Methods

Old C57Bl/6 mice (aged over 16 months) were intraperitoneally injectedwith 1 mg/kg of Neonatal Vaccine-induced IgG or the same amount ofisotyped matched IgG as a control daily for three days. 24 h after thelast dose the mice were challenged subcutaneously with 2×10⁷ CFU of GBSNEM316. Survival of the mice following infection was monitored on adaily basis for 12 days.

Two independent experiments were performed.

Results

Vaccination with Neonatal Vaccine was shown to protect old mice againstlethal GBS infection. Indeed, eight out of nine mice (˜90%) injectedwith Neonatal Vaccine survived the bacterial challenge compared to onlyone of ten (10%) controls (FIG. 20).

Discussion

This result shows that the vaccines of the invention, including NeonatalVaccine, can direct the immune system of old mice to a robust andspecific response towards sepsis-inducing agents, as exemplified by GBS,in a parallel fashion to that demonstrated in neonates (see Example 9).

The inventors firmly believe, therefore, that susceptibility toinfection by sepsis-inducing bacteria in the elderly is underpinned bythe same mechanism as they have discovered in neonates (i.e. GAPDH,which is secreted by the GBS bacteria, acts on Bi cells through TLR2 inorder to induce IL-10 production). Indeed, the data provided herein showthat Neonatal Vaccine can be used to produce antibodies that recognisebacterial GAPDH produced by GBS, and this is clearly having a protectiveeffect in the old mice, just as has been observed in neonates. Theinventors therefore also firmly believe that the same would be true forother such immunocompromised hosts.

Although this study only looked at GBS infection, it is understood thatthe same result would be observed upon infection by othersepsis-inducing bacteria (not least because Example 8 shows thatantibodies elicited with Neonatal Vaccine recognise GAPDH from GBSstrain NEM316, P. aeruginosa, S. pneumoniae, S. aureus, E. coli, K.pneumoniae and N. meningitidis (as exemplified by MenB)). The resultsseen with Neonatal Vaccine in neonatal mice challenged with thedifferent bacterial strains (see Example 10) could therefore reasonablybe expected in other immunocompromised hosts, such as old mice, too.

As described herein, currently there is no available vaccine thatefficiently protects the elderly against infections caused by any of themost relevant sepsis-inducing bacteria. Therapeutic strategies to combatsepsis in this group are also far from effective. Presented herein aredata demonstrating that antibody-mediated neutralisation of bacterialGAPDH prevents infections caused by the most relevant sepsis-inducingbacteria in the elderly. Vaccination is the most cost-effectivetreatment for infectious diseases, even more so when the same vaccinecould prevent infections caused by different human pathogens indifferent age groups, as has been demonstrated here.

The data obtained in the old mice are proof-of-concept that the otherresults obtained in the neonates would be obtained in the elderly andother such immunocompromised hosts too. The administration of NeonatalVaccine IgG antibodies to old mice suffering an existing infectioncaused by sepsis-inducing bacteria is therefore expected to result intheir treatment, just as has been observed in the neonates (see Example11). As the current treatment available for sepsis is based only onantibiotic administration, the fact that antibodies induced by NeonatalVaccine could be used to treat existing infections caused by the mostrelevant sepsis-inducing bacteria in the elderly, as well as in neonatesand the other patient populations indicated herein, is clearlyadvantageous.

Example 13—Neonatal Vaccine Protects NOD Mice Against GBS Infection

This study aimed to show that the antibodies produced in Example 8 couldbe used to protect a transgenic mouse model of diabetes (NOD mice) fromGBS infection.

Materials and Methods

NOD mice (eight weeks of age) were intraperitoneally injected with 1mg/kg of Neonatal Vaccine-induced IgG or the same amount of isotypedmatched IgG as a control daily for three days. 24 h after the last dosethe mice were challenged subcutaneously with 5×10⁷ CFU of GBS NEM316.Survival of the mice following infection was monitored on a daily basisfor 12 days.

Two independent experiments were performed.

Results

Passive immunisation using Neonatal Vaccine-induced IgG was shown toprotect NOD mice against lethal GBS infection. Indeed, seven out ofeight mice (˜90%) injected with Neonatal Vaccine-induced IgG survivedthe bacterial challenge compared to only two of eight (25%)sham-immunised controls (FIG. 21).

Discussion

This result shows that the vaccines of the invention, including NeonatalVaccine, can direct the immune system of a transgenic mouse model ofdiabetes to a robust and specific response towards sepsis-inducingagents, as exemplified by GBS, in a parallel fashion to thatdemonstrated in neonates (see Example 9) and the elderly (see Example12).

The inventors firmly believe, therefore, that susceptibility toinfection by sepsis-inducing bacteria in diabetics, as per the elderly,is underpinned by the same mechanism as they have discovered in neonates(i.e. GAPDH, which is secreted by the GBS bacteria, acts on B1 cellsthrough TLR2 in order to induce IL-10 production). Indeed, the dataprovided herein show that Neonatal Vaccine can be used to produceantibodies that recognise bacterial GAPDH produced by GBS, and this isclearly having a protective effect in the diabetic mice, just as hasbeen observed in neonates (and the elderly). The inventors thereforealso firmly believe that the same would be true for other suchimmunocompromised hosts.

Again, although this study only looked at GBS infection, it isunderstood that the same result would be observed upon infection byother sepsis-inducing bacteria (not least because Example 8 shows thatantibodies elicited with Neonatal Vaccine recognise GAPDH from GBSstrain NEM316, P. aeruginosa, S. pneumoniae, S. aureus, E. coli, K.pneumoniae and N. meningitidis (as exemplified by MenB)). The resultsseen with Neonatal Vaccine in neonatal mice challenged with thedifferent bacterial strains (see Example 10) could therefore reasonablybe expected in other immunocompromised hosts, such as diabetics, too.

As described herein, diabetic patients have increased susceptibility toinfection by sepsis-inducing bacteria. Presented herein are datademonstrating that antibody-mediated neutralisation of bacterial GAPDHprevents infections caused by the most relevant sepsis-inducing bacteriain this patient group. As explained, vaccination is the mostcost-effective treatment for infectious diseases, even more so when thesame vaccine could prevent infections caused by different humanpathogens in different age groups and across different diseases,conditions and disorders, as has been demonstrated here.

The data obtained in the diabetic mice are also proof-of-concept thatthe other results obtained in the neonates would be obtained indiabetics and other such immunocompromised hosts too. The administrationof Neonatal Vaccine IgG antibodies to diabetic mice suffering anexisting infection caused by sepsis-inducing bacteria is thereforeexpected to result in their treatment, just as has been observed in theneonates (see Example 11).

As the current treatment available for sepsis is based only onantibiotic administration, the fact that antibodies induced by NeonatalVaccine could be used to treat existing infections caused by the mostrelevant sepsis-inducing bacteria in diabetics, as well as in neonates,the elderly and other patient populations indicated herein, is clearlyadvantageous.

CONCLUDING REMARKS

The data presented in the Examples show the relevance of vaccines andtreatments of the invention, including Neonatal Vaccine, to protectimmunocompromised hosts such as neonates, babies, children, women offertile age, pregnant women, foetuses and the elderly, in particular,from bacterial sepsis.

Moreover, the rationale of Neonatal Vaccine and the other vaccines andtreatments described herein represents significant new inventive stepsregarding the previous published results [24,25], namely:

a) The mechanism by which bacterial GAPDH induces IL-10 in the neonatalhost;b) GAPDH-induced IL-10 production is associated with susceptibility tobacterial sepsis caused by different pathogens;c) GAPDH-induced IL-10 production is a mechanism conserved in humancord-blood cells;d) GAPDH-induced IL-10 production is a mechanism conserved in leukocytesisolated from the peripheral blood of adult humans;e) The efficacy of anti-GAPDH antibodies in preventing stillbirthscaused by GBS;f) Antibodies elicited by Neonatal Vaccine (and other vaccines describedherein) recognise extracellular GAPDH from GBS, P. aeruginosa, E. coli,S. pneumoniae, K. pneumoniae, S. aureus and N. meningitidis.g) Neutralisation of bacterial GAPDH by means of passive immunisationwith antibodies elicited with Neonatal Vaccine (and other vaccinesdescribed herein), protects newborns from sepsis caused by GBS, E. coli,S. pneumoniae and S. aureus;h) The use of peptides derived from GAPDH of sepsis-inducing bacteriaand the use of anti-GAPDH IgG antibodies, either as a preventivestrategy or as a treatment for neonatal sepsis and sepsis in otherpatient groups as indicated herein.

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1. An isolated peptide that has at least 90% amino acid sequenceidentity with a peptide found within GAPDH of one or moresepsis-inducing bacteria, and has less than 10% amino acid sequenceidentity with a peptide found within human GAPDH, or a functionalfragment or functional variant thereof, wherein the peptide is less than50 amino acids in length.
 2. An isolated peptide, fragment or variantthereof as claimed in claim 1, wherein the peptide has at least 90%amino acid sequence identity with a peptide found within GAPDH of GBS,E. coli, Staphylococcus spp., S. pneumoniae, K. pneumoniae, N.meningitidis and/or Pseudomonas spp.
 3. An isolated peptide, fragment orvariant thereof as claimed in claim 1 or claim 2, wherein the peptidehas at least 95%, at least 98%, at least 99% or 100% amino acid sequenceidentity with said peptide found within GAPDH of sepsis-inducingbacteria.
 4. An isolated peptide, fragment or variant thereof as claimedin any one of the preceding claims, wherein the peptide has an aminoacid sequence substantially as set out in any one of SEQ ID NOs: 9-69.5. An isolated peptide, fragment or variant thereof as claimed in anyone of the preceding claims, wherein the peptide: a) comprises less than30 amino acids or less than 20 amino acids; b) comprises at least 3amino acids, at least 5 amino acids, at least 8 amino acids or at least10 amino acids; and/or c) is 5-50 amino acids in length or 10-20 aminoacids in length.
 6. An isolated nucleic acid encoding a peptide,fragment or variant thereof as claimed in any one of the precedingclaims, or a functional fragment or functional variant thereof.
 7. Agenetic construct comprising a nucleic acid, fragment or variant thereofas claimed in claim
 6. 8. A recombinant vector comprising a geneticconstruct as claimed in claim
 7. 9. A host cell comprising a geneticconstruct as claimed in claim 7, or a recombinant vector as claimed inclaim
 8. 10. A transgenic host organism comprising at least one hostcell as claimed in claim
 9. 11. Use of a peptide, fragment or variant asclaimed in any one of claims 1-5, in the development of a vaccine forpreventing an infection with sepsis-inducing bacteria.
 12. A vaccinecomprising a peptide, fragment or variant as claimed in any one ofclaims 1-5.
 13. A vaccine as claimed in claim 12, comprising two or morepeptides, fragments or variants as claimed in any one of claims 1-5,wherein at least two of the peptides, fragments and/or variants arelinked together.
 14. A vaccine as claimed in claim 12 or claim 13,comprising one or more peptides having an amino acid sequencesubstantially as set out in SEQ ID NOs: 9-69.
 15. A vaccine as claimedin any one of claims 12-14, comprising any one, two, three or all fourof the peptides having the amino acid sequences as set out in SEQ IDNOs: 9-12.
 16. A vaccine as claimed in any one of claims 12-15,formulated for administration to an immunocompromised host preferablyselected from a neonate, baby, child, woman of fertile age, pregnantwoman, foetus, diabetic and/or an elderly subject.
 17. Use of a peptide,fragment or variant as claimed in any one of claims 1-5, or a vaccine asclaimed in any one of claims 12-16, for stimulating an immune response.18. A use as claimed in claim 17, wherein the immune response includesthe production of antibodies that are specific to GAPDH of one or morespecies of sepsis-inducing bacteria.
 19. A use as claimed in claim 17 orclaim 18, wherein the use is: a) an in vitro, in vivo or ex vivo use;and/or b) an in vitro or ex vivo use for stimulating the production ofmonoclonal or polyclonal antibodies.
 20. A peptide, fragment or variantas claimed in any one of claims 1-5, for use in therapy.
 21. A peptide,fragment or variant as claimed in any one of claims 1-5, or a vaccine asclaimed in any one of claims 12-16, for use in preventing an infectionby sepsis-inducing bacteria.
 22. A peptide, fragment or variant asclaimed in any one of claims 1-5, or a vaccine as claimed in any one ofclaims 12-16, for use in preventing any one or more of sepsis,pneumonia, meningitis, endocarditis, enterocolitis, urinary tractinfections, soft tissue infections, gastrointestinal infections,bloodstream infections, encephalitis, premature birth and stillbirth.23. A method of preventing an infection by sepsis-inducing bacteria, themethod comprising administering, to a subject in need of such treatment,a peptide, fragment or variant as claimed in any one of claims 1-5 or avaccine as claimed in any one of claims 12-16.
 24. A method as claimedin claim 23, wherein the method is a method of preventing any one ormore of sepsis, pneumonia, meningitis, endocarditis, enterocolitis,urinary tract infections, soft tissue infections, gastrointestinalinfections, bloodstream infections, encephalitis, premature birth andstillbirth.
 25. An antibody that is specific for GAPDH of one or morespecies of sepsis-inducing bacteria, said antibody being raised againsta peptide, fragment or variant as claimed in any one of claims 1-5 or avaccine as claimed in any one of claims 12-16.
 26. An antibody that isspecific for an epitope found in GAPDH of one or more species ofsepsis-inducing bacteria, wherein the epitope has an amino acid sequencesubstantially as set out in any one of SEQ ID NOs: 9-69.
 27. An antibodyas claimed in claim 26, wherein the antibody is raised against apeptide, fragment or variant as claimed in any one of claims 1-5 or avaccine as claimed in any one of claims 12-16.
 28. An antibody asclaimed in any one of claims 25-27, for use in therapy.
 29. An antibodyas claimed in any one of claims 25-27, for use in preventing, treatingor ameliorating an infection by sepsis-inducing bacteria.
 30. Anantibody as claimed in any one of claims 25-27, for use in preventing,treating or ameliorating any one or more of sepsis, pneumonia,meningitis, endocarditis, enterocolitis, urinary tract infections, softtissue infections, gastrointestinal infections, bloodstream infectionsand encephalitis, and/or for use in preventing premature birth and/orstillbirth.
 31. A method of producing antibodies that are specific forGAPDH of one or more species of sepsis-inducing bacteria, the methodcomprising the step of contacting antibody-producing cells with apeptide, fragment or variant as claimed in any one of claims 1-5, or avaccine as claimed in any one of claims 12-16.
 32. A method as claimedin claim 31, wherein the method is: a) an in vitro, in vivo or ex vivomethod; b) an in vitro or ex vivo method of producing monoclonal orpolyclonal antibodies; c) an in vivo method of vaccination.
 33. A methodof treating, ameliorating or preventing an infection by sepsis-inducingbacteria, the method comprising administering, to a subject in need ofsuch treatment, an antibody as claimed in any one of claims 25-27.
 34. Amethod as claimed in claim 33, wherein the method is a method oftreating, ameliorating or preventing any one or more of sepsis,pneumonia, meningitis, endocarditis, enterocolitis, urinary tractinfections, soft tissue infections, gastrointestinal infections,bloodstream infections and encephalitis, and/or is a method ofpreventing premature birth and/or stillbirth.
 35. A method as claimed inclaim 23 or claim 33, wherein the subject in need of treatment is animmunocompromised host preferably selected from a neonate, baby, child,woman of fertile age, pregnant woman, foetus, diabetic and/or an elderlysubject.
 36. A sepsis-inducing bacteria treatment composition comprisingan antibody as claimed in any one of claims 25-27, and optionally apharmaceutically acceptable vehicle.
 37. A process for making acomposition as claimed in claim 36, the process comprising combining atherapeutically effective amount of an antibody as claimed in any one ofclaims 25-27, with a pharmaceutically acceptable vehicle.
 38. A use asclaimed in claim 11 or claim 18, a peptide, fragment or variant asclaimed in claim 21, a method as claimed in claim 23, claim 31 or claim33, an antibody as claimed in any one of claims 25, 26 or 29, or acomposition as claimed in claim 36, wherein the sepsis-inducing bacteriaare selected from the group consisting of GBS, E. coli, Staphylococcusspp., S. pneumoniae, K. pneumoniae, Pseudomonas spp. and N.meningitidis.